Full-length haplotype reconstruction of CD36 by long-read sequencing: uncovers a novel structural variant

Full-length haplotype reconstruction of CD36 by long-read sequencing: uncovers a novel structural variant | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Full-length haplotype reconstruction of CD36 by long-read sequencing: uncovers a novel structural variant Shuang Liang, Tong Liu, Huatao Che, Wenxia Xia, Weiyi Fu, Fan Wu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8004674/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in BMC Genomics → Version 1 posted 11 You are reading this latest preprint version Abstract Purpose: CD36 deficiency predisposes to fetal/neonatal alloimmune thrombocytopenia (FNAIT) and platelet transfusion refractoriness (PTR), yet its genetic architecture remains incompletely understood. This study employed long-read sequencing to reconstruct full-length CD36 haplotypes, aiming to identify its genetic backgrounds. Methods: We developed a long-read sequencing approach to reconstruct full-length CD36 haplotypes from four overlapping amplicons. Samples from 43 CD36-deficient individuals (14 type I, 29 type II) were analyzed. Structural variants were validated via PacBio whole-genome sequencing, platelet CD36 expression was quantified by flow cytometry, and a gap-PCR assay screened 600 blood donors for large deletions. Results: Full-length 77-kb CD36 haplotypes were reconstructed for the first time using overlapping amplicons. All 28 haplotypes from type I deficiency samples harbored known or novel variations, including a novel structural variant (c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA, 19,971 bp deletion-insertion) spanning intron 1-3 that eliminates exons 2/3 and the translation initiation site. Among 58 haplotypes from type II cases, pathogenic variants were identified in only 26/58 (44.8%) (including one haplotype carrying the novel structural variant), while 32/58 (55.2%) lacked detectable variations. Gap-PCR screening revealed a 0.50% carrier frequency for this structural variant in blood donors; all heterozygotes exhibited wild-type CD36 on the alternate allele with normal platelet CD36 expression confirmed by flow cytometry. Conclusions: A long-read sequencing approach with four overlapping amplicons was developed to successfully reconstruct the full-length CD36 haplotypes, which overcamelimitations of conventional genotyping and provided a robust technical foundation for identifying structural variants. CD36 Full-length haplotype Long-Read Sequencing Structural variant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Points A long-read sequencing approach was established and successfully and firstly reconstructed the complete ~77kb CD36 haplotype. CD36-deficient donors included a novel 19,971bp deletion-insertion structural variant without antigen dosage effect of heterozygotes. 1. Introduction CD36, also known as glycoprotein IV (GPIV), is an 88-kDa transmembrane protein expressed on a wide range of cell types including platelets, monocytes, endothelial cells, and erythroid precursors, though expression on mature red blood cells (RBCs) is relatively low 1 , 2 . Initially identified as a platelet membrane component, CD36 has since been implicated in diverse physiological functions including lipid metabolism, innate immunity, and cell adhesion, and it also serves as a key antigen target in alloimmune thrombocytopenia disorders 3 – 5 . CD36 deficiency is classified into two phenotypes: type I, characterized by complete absence of expression on both platelets and monocytes, and type II, defined by selective absence on platelets 6 , 7 . Although individuals with CD36 deficiency are often clinically asymptomatic, exposure to CD36-positive cells through blood transfusion or pregnancy can lead to alloimmunization and the production of anti-CD36 antibodies 8 , 9 . These antibodies are known to cause serious immune-mediated complications such as FNAIT, PTR, and posttransfusion purpura 8 , 10 – 12 . Importantly, CD36-negative phenotypes vary in prevalence across populations, reaching up to 11% in certain Asian cohorts, and have become increasingly relevant to transfusion practice in regions with active donor surveillance 13 , 14 . The CD36 gene, located on chromosome 7q21.11, spans approximately 77 kb and includes 15 exons, 12 of which are coding 15 . Although over 30,000 variants are listed in the dbSNP database, only a small subset is known to affect protein expression 16 . Most studies have focused on coding variants and selected single nucleotide variants (SNVs), while the broader haplotype structure and upstream regulatory regions remain understudied 14 , 17 – 18 . Using PacBio sequencing, Xia et al. obtained ~ 40 kb haplotypes from exon 2 to the 3’ untranslated region (UTR) 19 . However, in some type I deficiency cases, causative variants could not be identified within these regions, suggesting that critical regulatory elements might reside in intron 1 or adjacent noncoding regions 20 , 21 . In this study, we used four overlapping long-range polymerase chain reaction (PCR) amplicons (20 ~ 23 kb each) to generate full-length (~ 77 kb) haplotype sequences of the CD36 gene using the Polyseq nanopore sequencing platform 22 . Applying this approach, we analyzed samples from 14 individuals with type I CD36 deficiency and 29 with type II deficiency. In three type I and one type II CD36 deficiency samples, we detected potential structural variants located in intron 1. Among them, two cases were further validated by PacBio whole-genome sequencing, which confirmed a 19,971 bp deletion (c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA) with precise breakpoint resolution. Finally, we screened 300 routine blood donors using a PCR-based assay and estimated the allele frequency of this large deletion haplotype to be approximately 0.6%. 2. Methods and Materials 2.1 Sample Collection Fourteen type I and twenty-nine type II CD36-deficient samples were randomly selected from the Shenzhen Blood Center CD36 Deficiency Bank. An additional 600 normal samples were collected from routine healthy donors at the same center. Peripheral blood (5.0 mL) was collected in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes for genomic DNA extraction and flow cytometry analysis. A graphical overview of this study is provided in Fig. 1 . All donors were of Han ethnicity and all samples were obtained with informed consent under ethical approval from the Shenzhen Blood Center Ethics Committee (SZBCMEC-2024-047). 2.2 Genomic DNA Extraction Genomic deoxyribonucleicacid (DNA) was isolated from EDTA-anticoagulated peripheral blood using the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics). DNA purity (A₂₆₀/A₂₈₀ ratio 1.8-2.0) and concentration were assessed by spectrophotometry (NanoDrop 8000), and extracts were normalized to 30 ng/µL in Tris-HCl/EDTA buffer for downstream use. 2.3 LR-PCR for Full-Length CD36 Gene Primer sets ( CD36 1Forward/Reverse-4 Forward/Reverse) were designed to amplify regions spanning from the 5’ UTR to the 3’ UTR of the CD36 gene, covering a total length of approximately 77 kb and including selected intronic sequences. Four sets were developed to generate amplicons ranging from 21–23 kb in length, with ≥ 3 kb overlap between adjacent fragments (Fig. 2 ). All primer sequences are listed in Table 1 . Long-range PCR (LR-PCR) was performed using ApexHF HS DNA Polymerase CL (Accurate Biology, AG12204) according to the manufacturer’s instructions. PCR cycling consisted of a three-step program: 98°C for 10 s, 65°C for 10 s, and 72°C for 3.5 min, for 30 cycles. Table 1 Primer pairs used for full-length CD36 gene amplification and gap-PCR assay. Primer Sequence (5’→3’) Size Coverage Usage CD36 1-F ctccctcaccacctatccctataagct 20.6 kb 5’UTR ~ exon 1 LR-PCR primers for LRS CD36 1-R ctgccactgcttcatcaaccaacttta CD36 2-F caaagtggctacggcgattgtaaggatc 22 kb intron 1 CD36 2-R acagtagtgtcacctcccgtcatctgg CD36 3-F tccaaattcagactcggattccattcat 20.6 kb exon 2 ~ 3 CD36 3-R accttaccatacacttgcagcagaaat CD36 4-F ggcttagacagtaaatgctatgaaccaag 23 kb exon 4 ~ 14 CD36 4-R caccgtcttacactttgctacttcttaca CD36 5-F GTGTCAAACTGTCATCGGGGTG 20 kb exon 2 ~ 3 deletion-flanking primers for gap-PCR CD36 5-R GACCCTTTTCCACAGTGCTTTG CD36 6-F CAGAGAGATGTGGAGGGGAGTT 290 bp intron 7 control primers for gap-PCR CD36 6-R TCTTGCCCCTTCCTCCCTTTA Note : F: forward, R: reverse. 2.4 Library Preparation, Nanopore Sequencing and Bioinformatic Analysis Library preparation was performed using the commercial PY-DTB101/102 and PY-BLP101 kits (Polyseq Biotech Co. Ltd., China) according to the manufacturer’s instructions. Prepared libraries were loaded onto PY-NFC001 flow cells and sequenced for 12 hours on the PolyseqOne nanopore sequencing platform ( www.polyseq.com ). Basecalling and demultiplexing were performed using Kant v1.0.1 in high-accuracy mode (processing rate: 420 bases per second). Raw reads underwent demultiplexing with automated barcode identification. For CD36 sequence analysis, reads were first aligned to the T2T-CHM13v2.0 reference genome and error-corrected using NanoFix-AI (DAFEI Biotechnology, Guangzhou, China) 23 . FASTQ reads corresponding to individual CD36 alleles were extracted using bedtools (v2.30.0). A custom Python workflow was then employed to convert these reads into clustered BAM files. Finally, the clustered reads were re-aligned to the CD36 reference gene sequence using pbmm2. 2.5 Whole-Genome Sequencing Analysis Long-read whole-genome sequencing was performed on the PacBio Revio platform (Pacific Biosciences), generating 31.2 Gb and 52.4 Gb of HiFi reads with median qualities of Q35. Coverage depth across the CD36 gene was 25× and 41×, respectively. Reads were aligned to the human reference genome (CHM13v2.0) using minimap2 (v2.14-r883) with default parameters. Large deletions were detected using software developed by DAFEI Biotechnology (Guangzhou, China). Coverage was assessed with mosdepth, and deletion breakpoints/junctions were manually verified using the Integrative Genomics Viewer (IGV). CD36 sequence data were visualized in SnapGene for clear presentation of structural findings. 2.6 Population Screening for 19,971-bp Deletion-Insertion Structural Variant We developed a multiplex gap-PCR assay to screen 600 blood donors for the recurrent 19,971bp deletion-insertion structural variant spanning exons 2–3. Two primer pairs were designed based on the CD36 gene sequence (GenBank ID: NG_008192.1): control primers ( CD36 6F/R) amplifying a 333-bp fragment in intron 7, and deletion-flanking primers ( CD36 5F/R) positioned 571 bp upstream (5F) and 261 bp downstream (5R) of the deletion boundaries. All primer sequences are listed in Table 1 . PCR amplification was performed in multiplex format under the following conditions: 30 cycles of 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min, with pre-validated positive and negative controls included in each run. All candidate positives underwent confirmation through nanopore sequencing (Polyseq platform) to verify breakpoint identity with whole-genome sequencing data and determine zygosity status. 2.7 Flow cytometric analysis for CD36 Expression on Platelets CD36 surface expression on platelets was assessed by flow cytometry using fresh whole blood samples processed within 24 hours of collection. Platelet-rich plasma (PRP) was isolated from EDTA-anticoagulated blood by centrifugation at 200 ×g for 10 min at room temperature. Following three washes with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 1 mM EDTA (PBS-BSA-EDTA), platelets were resuspended at 1×10⁷ cells/ml. For staining, 20 µL platelet suspension was incubated with 2 µL FITC-conjugated anti-CD36 antibody (clone FA6-152; BD Biosciences, USA) for 20 min at room temperature in the dark. After three washes with PBS-BSA-EDTA, cells were resuspended in 500 µL PBS. Analysis was performed on a FACSCanto II flow cytometer (BD Biosciences) with platelets gated by characteristic forward/side scatter (FSC/SSC) profiles. CD36 expression was quantified as mean fluorescence intensity (MFI) using FACSDiva software v8.0.1 (BD Biosciences). 3. Results 3.1 Reconstruction of Full-Length CD36 Haplotypes by Nanopore Sequencing Full-length (~ 77 kb) CD36 haplotypes were reconstructed by SNP-based phasing of four overlapping 21–23 kb amplicons, sequenced using the Polyseq nanopore platform (Fig. 2 ). Using this approach, we analyzed samples from 14 type I and 29 type II CD36-deficient individuals. All identified exonic variants (including those within 10 bp of exon-intron boundaries) present on each haplotype are summarized in Table 2 . This represents the first successful reconstruction of the complete ~ 77 kb CD36 haplotype, providing a comprehensive view of its genetic architecture. Table 2 Genetic variations in CD36 exons among haplotype sequences from CD36 deficient samples. Sample ID Haplotype 1 Haplotype 2 Type I-01 c.-132A > C c.430-1G > C; c.-132A > C I I-02 c.-132A > C c.1228_1239delATTGTGCCTATT I I-03 c.-132A > C c.1229T > C*; c.-132A > C I I-04 c.332_333delCA; c.-132A > C c.1228_1239delATTGTGCCTATT I I-05 c.332_333delCA; c.-132A > C c.1228_1239delATTGTGCCTATT I I-06 c.332_333delCA; c.-132A > C c.1228_1239delATTGTGCCTATT I I-07 c.380C > T c.1228_1239delATTGTGCCTATT I I-08 c.430-2A > G c.609 + 7A > G*; c.-132A > C I I-09 c.1228_1239delATTGTGCCTATT c.1156C > T I I-10 c.1228_1239delATTGTGCCTATT c.1156C > T; c.1409C > T I I-11 c.1228_1239delATTGTGCCTATT c.1228_1239delATTGTGCCTATT I I-12 c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA c.1006 + 2T > G I I-13 c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA c.1156C > T; c.1409C > T I I-14 c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA c.1163A > T I II-01 c.-132A > C - II II-02 c.-132A > C - II II-03 c.220C > T; c.-132A > C c.-132A > C II II-04 c.275C > T - II II-05 c.332_333delCA - II II-06 c.332_333delCA; c.-132A > C - II II-07 c.332_333delCA; c.-132A > C - II II-08 c.332_333delCA; c.-132A > C - II II-09 c.332_333delCA; c.-132A > C - II II-10 c.332_333delCA; c.-132A > C - II II-11 c.429 + 3insG* - II II-12 c.847G > A - II II-13 c.1156C > T - II II-14 c.1156C > T - II II-15 c.1156C > T; c.1409C > T - II II-16 c.1228_1239delATTGTGCCTATT - II II-17 c.1228_1239delATTGTGCCTATT - II II-18 c.1228_1239delATTGTGCCTATT c.-132A > C II II-19 c.1228_1239delATTGTGCCTATT c.609 + 7A > G*; c.-132A > C II II-20 c.1228_1239delATTGTGCCTATT; c.-132A > C - II II-21 c.1340_1343dupTCTT - II II-22 c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA c.681C > A* II II-23 - - II II-24 - - II II-25 - - II II-26 - - II II-27 - - II II-28 - - II II-29 - - II *Represents the new variants identified in this study. 3.2 Variant Spectrum and Structural Variants in Type I and II CD36 Deficiency Analysis of the haplotypes derived from 14 type I deficiency samples revealed that all harbored either previously reported pathogenic point variations or complex structural variants, including 13 compound heterozygous variants and 1 homozygous variant. The most frequent variant was c.-132A > C and c.1227delTATTGTGCCTAT, both detected in 32.1% (9/28) of haplotypes. The remaining 35.7% (10/28) of haplotypes harbored a spectrum of other variants, including the novel 19,971-bp deletion-insertion structural variant (10.7%, 3/28) and multiple other point variations and indels as detailed in Table 2 . In contrast, among the 28 type II deficiency samples, 22 exhibited variants in the CD36 gene (18 compound heterozygous variants and 4 heterozygous variants), the predominant variant was c.-132A > C, identified in 20.7% (12/58; assuming a comparable denominator context, or specify total type II haplotypes if known). Thus, we successfully obtained complete haplotype sequences in 43 CD36 deficiency samples using the Polyseq nanopore platform, enabling phased variant calling that was not achievable by previous methods. All haplotype sequences have been submitted to the NCBI database (Table 3 ). Table 3 Genetic variations of the CD36 gene in CD36 deficient samples. Genetic variations Exon/Intron Type I deficiency (n = 28) Type II deficiency (n = 58) Accession no. n Frequency n Frequency c.220C > T exon 4 0 0.00% 1 1.67% PV866985 c.275C > T exon 4 0 0.00% 1 1.67% PV866988 c.332_333delCA exon 5 3 10.00% 6 10.00% PV808479 c.380C > T exon 5 1 3.33% 0 0.00% PV808480 c.429 + 3insG intron5 0 0.00% 1 1.67% PV866987 c.430-1G > C intron5 1 3.33% 0 0.00% PV866984 c.430-2A > G intron5 1 3.33% 0 0.00% PV832518 c.609 + 7A > G intron6 1 3.33% 1 1.67% PV808482 c.681C > A exon 7 0 0.00% 1 1.67% PV855215 c.847G > A exon 10 0 0.00% 1 1.67% PV866986 c.1006 + 2T > G intron10 1 3.33% 0 0.00% PV866989 c.1156C > T exon 12 1 3.33% 2 3.33% PV808481 c.1156C > T; c.1409C > T exon 12, 14 2 6.67% 1 1.67% PV855214 c.1163A > T exon 12 1 3.33% 0 0.00% PV855217 c.1227delTATTGTGCCTAT exon 13 9 30.00% 5 8.33% PV786778 c.1229T > C exon 13 1 3.33% 0 0.00% PV855218 c.1340_1343dupTCTT exon 14 0 0.00% 1 1.67% PV855216 c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA exon 2–3 3 10.00% 1 1.67% PV866990 3.3 A Novel Structural Variant Spanning CD36 Exons 2–3 In four CD36-deficient samples (3 type I, 1 type II), nanopore sequencing revealed consistent amplification failure of amplicons 2–3 in one haplotype (Fig. 3 A), suggesting structural variants. To further characterize this inspection, we performed whole-genome sequencing on two deficient samples (I-13 and II-22) using the PacBio Revio platform. This generated 31.2 Gb and 52.4 Gb of HiFi reads, respectively, with a median quality score of Q35; the majority of reads exceeded 10 kb in length (Figure S1 ). Visualization using IGV revealed a sharp drop in coverage and disrupted alignment across the second half of intron 1 (Fig. 3 B). Subsequent de novo assembly identified a 19,971 bp deletion-insertion structural variant (c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA, NG_008192.1: g.33588_g.53560delinsCCAATGCTAAGGTTGA) in one haplotype of each sample. This deletion spans positions c.1-15966 (following a 16-bp insertion) to c.120 + 3887 (relative to the CD36 reference sequence, Fig. 3 C), thereby removing exons 2 and 3, including the translation initiation site located in exon 3. 3.4 Population Screening for 19,971-bp Deletion-Insertion Structural Variant Under the established gap-PCR conditions, amplification of the wild-type allele (20.8-kb) consistently failed, whereas the deletion allele yielded a distinct 832-bp fragment. Consequently, samples exhibiting both the 333-bp control and 832-bp bands were classified as potential carriers (Fig. 4 A). Screening of 600 blood donors using this assay identified three heterozygous carriers (Fig. 4 B), indicating a population carrier frequency of 0.50% (3/600), and the full uncropped Gels and Blots image(s) were displayed as “Supplementary file”. Nanopore sequencing confirmed that the breakpoints in these carriers were identical to those observed in the CD36-deficient cases (Fig. 5 A). Critically, flow cytometric analysis demonstrated preserved CD36 antigen expression on platelets in all three heterozygotes (Fig. 5 B), indicating that the deletion alone does not cause haploinsufficiency. 4. Discussion CD36 deficiency poses a significant risk in transfusion medicine due to its potential to induce alloantibodies that can cause PTR and FNAIT, particularly in Asian and African populations where its prevalence is higher 5 – 7 . Despite its clinical importance, the genetic architecture of CD36 deficiency remains incompletely understood. Although multiple point variations and small indels in exonic and splice-site regions have been reported, these do not fully explain the observed phenotypic variation 8 , 10 , 11 , 14 . Evidence suggests that structural variants and regulatory elements in noncoding regions may contribute to the pathogenesis, especially in type I deficiency 12 , 15 . Additionally, the highly polymorphic nature of the CD36 locus complicates the establishment of clear genotype-phenotype correlations 13 . In this study, we applied long-read sequencing to reconstruct full-length CD36 haplotypes, achieving comprehensive resolution of the ~ 77 kb locus for the first time. By integrating the previously reported c.-132A > C promoter variant and c.1228-1239delATTGTGCCTATT deletion variant 14 , as well as the novel 19,971-bp deletion-insertion structural variant identified here, we successfully explained the molecular basis of all 28 type I deficiency haplotypes. Compared with variants were detected in all haplotypes of type I deficiency samples, no CD36 gene variants were detected in either haplotype in 7 type II deficiency samples. Furthermore, although I-02 and II-18 had the same allele genotype (c.1228_1239delATTGTGCCTATT on one allele and c.-132A > C on the other), their CD36 antigen expression levels exhibit significant differences. All of the above suggesting that other mechanisms-such as regulatory element alterations, aberrant splicing, or epigenetic changes-may underlie this phenotype 24 , 25 . Our amplicon-based sequencing strategy provides a foundation for further investigation into these unresolved cases. The discovery of the 19,971-bp deletion within CD36 carries critical implications for molecular diagnostics. Our identification of the structural variant, which removes the translation initiation site in exon 3, explains its severe impact on protein expression when homozygous or compound heterozygous. Importantly, this structural variant was undetectable by Sanger sequencing in all four cases where it occurred, as conventional methods failed to amplify across the breakpoints localized deep within introns 1 and 3. This diagnostic gap likely led to prior misclassification of compound heterozygotes as homozygotes for point variations (e.g., in exons 4–14), underscoring a fundamental limitation of targeted exon sequencing. Population screening revealed a carrier frequency of 0.50% for this deletion. Heterozygous individuals exhibited normal CD36 expression, indicating that the variant does not cause haploinsufficiency. However, compound heterozygosity, where this structural variant coexists with another pathogenic variant on the alternate allele, can manifest the CD36-deficient phenotype. The asymptomatic carriage of this structural variant in the general population carries important practical implications: implementing targeted screening for CD36-negative platelet donors in regions with high allele prevalence may provide an effective preventive strategy to mitigate alloimmunization risks 26 , 27 . Moreover, our long-read sequencing approach overcomes critical limitations of conventional genotyping methods 28 , 29 . Previous Sanger sequencing of exonic regions, while capable of identifying point variations and small indels, could not resolve the phased haplotype structure of CD36 30 . This frequently led to diagnostic ambiguities, such as the misinterpretation of hemizygous large deletions as homozygous point variations due to amplification failure of the deleted allele. Our method enables unambiguous phasing and accurate detection of structural variants, offering a more reliable molecular diagnostic framework for CD36 deficiency. In summary, this study resolves a major diagnostic gap in CD36 genotyping by demonstrating the utility of long-read sequencing for comprehensive haplotype analysis. Our findings support the integration of population screening and advanced genomic technologies into clinical practice to better manage transfusion-related risks associated with CD36 deficiency. 5. Conclusion This study establishes the first full-length haplotype map of the CD36 gene (~ 77 kb) through long-read sequencing of four overlapping amplicons. This approach enabled the discovery of a novel recurrent 19,971-bp deletion-insertion structural variant (c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA) spanning intron 1 to intron 3, which eliminates exons 2–3 and the translation initiation site. The deletion was identified in three type I and one type II deficiency haplotypes. Population screening of blood donors revealed a 0.50% allele frequency, with flow cytometry confirming normal CD36 expression on platelets in all heterozygous carriers. These findings resolve a critical diagnostic blind spot for structural variants undetectable by conventional methods and provide essential insights for clinical management of CD36-related alloimmune risks in transfusion medicine. Abbreviations FNAIT - Fetal/Neonatal Alloimmune Thrombocytopenia PTR - Platelet Transfusion Refractoriness GPIV - Glycoprotein IV RBCs - Red Blood Cells SNV – Single Nucleotide Variant UTRs –Untranslated Regions PCR - Polymerase Chain Reaction DNA - Deoxyribonucleicacid EDTA - Ethylenediaminetetraacetic Acid LR-PCR - Long-range PCR PRP - Platelet-Rich Plasma PBS - Phosphate Buffered Saline BSA - Bovine Serum Albumin MFI - Fluorescence Intensity Declarations Declaration of competing interest The authors declare that they have no competing interests. Ethics Approval and Consent This project was conducted in accordance with the guidelines of the Helsinki Declaration, and approved by the Shenzhen Blood Center Ethics Committee (SZBCMEC-2024-047). All involved participants provided written informed consent. Consent for Publication Not applicable. Data Availability The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0007933 (https://db.cngb.org/data_resources/project/CNP0007933/). The original data and materials from this research are available from the corresponding author upon reasonable request. Acknowledgements We thank the blood donors and patients in this study. Funding This work was supported by the Natural Science Foundation of Shenzhen Municipality (No. JCYJ20230807154000002), Guangdong Medical Foundation (No. B2025582), Shenzhen Key Medical Discipline Construction Fund (No. SZXK070) and Sanming Project of Medicine in Shenzen Municipality (No. SZSM202311032), Key Research and Development Plan of Department of Science and Technology of Zhejiang Province (No. 2024C03156). Authors’ Contributions Shuang Liang: Original draft, Investigation, Funding acquisition. Tong Liu: Methodology, Data curation. Huatao Che: Methodology, Formal analysis. Wenxia Xia: Methodology, Data curation. Weiyi Fu: Data acquisition. Fan Wu: Methodology. Liyan Sun: Methodology, Statistical analyses. Yongshui Fu: Writing-review ＆ editing. Faming Zhu: Writing-review ＆ editing. Dawei Cui: Writing-review ＆ editing, Supervision, Funding acquisition. All the authors have read and approved the final version of the manuscript. References van Schravendijk MR, Handunnetti SM, Barnwell JW, et al. Normal human erythrocytes express CD36, an adhesion molecule of monocytes, platelets, and endothelial cells. Blood . 1992;80(8):2105-2114. Tian J, Liu W, Gao W, et al. Molecular cloning and gene/protein expression of FAT/CD36 from grass carp (Ctenopharyngodon idella) and the regulation of its expression by dietary energy. Fish Physiol Biochem . 2017;43(3):875-888. Chen Y, Zhang J, Cui W, et al. CD36, a signaling receptor and fatty acid transporter that regulates immune cell metabolism and fate. J Exp Med. 2022;219(6):e20211314. Tzeng HT, Chyuan IT, Chen WY. Shaping of Innate Immune Response by Fatty Acid Metabolite Palmitate. Cells . 2019. 8(12):1633. Caruso JA, Wang X, Murrow LM, et al. Loss of PPARγ activity characterizes early protumorigenic stromal reprogramming and dictates the therapeutic window of opportunity. Proc Natl Acad Sci U S A . 2023;120(42):e2303774120. Masuda Y, Tamura S, Matsuno K, et al. Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects. Thromb Res. 2015;135(5):951-957. Ono R, Miyauchi H, Kobayashi Y. CD36 deficiency. QJM: An International Journal of Medicine . 2025;118(5):363-364. Wu Y, Chen D, Xu X, et al. Hydrops fetalis associated with anti‐CD36 antibodies in fetal and neonatal alloimmune thrombocytopenia: possible underlying mechanism. Transfus Med . 2020;30(5):361-368. Schmidt AE, Sahai T, Refaai MA, et al. Severe Platelet Transfusion Refractoriness in Association with Antibodies Against CD36. Lab Med . 2020;51(5):540-544. Khatri SS, Curtis BR, Yamada C. A case of platelet transfusion refractoriness due to anti-CD36 with a successful treatment outcome. Immunohematology . 2019;35(4):139-144. Xu X, Li L, Xia W, et al. Successful management of a hydropic fetus with severe anemia and thrombocytopenia caused by anti-CD36 antibody. Int J Hematol . 2018;107(2):251-256. Chen D, Liang H, Xu X, et al. Inhibition of terminal complement complex formation alleviates murine antibody-mediated TRALI. Blood . 2025;146(6):759-764. Masuda Y, Tamura S, Matsuno K, et al. Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects. Thromb Res . 2015;135(5):951-957. Kashiwagi H, Tomiyama Y, Kosugi S, et al. Identification of molecular defects in a subject with type I CD36 deficiency. Blood . 1994;83(12):3545-3552. Rać ME, Safranow K, Poncyljusz W. Molecular basis of human CD36 gene mutations. Mol Med . 2007;13(5-6):288-296. Masuda Y, Tamura S, Matsuno K, et al. Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects. Thromb Res . 2015;135(5):951-957. Xu X, Liu Y, Hong X, et al. Variants of CD36 gene and their association with CD36 protein expression in platelets. Blood Transfus . 2014;12(4):557-564. Madan N, Ghazi AR, Kong X, et al. Functionalization of CD36 cardiovascular disease and expression associated variants by interdisciplinary high throughput analysis. PLoS Genet . 2019;15(7):e1008287. Xia W, Chen D, Li X, et al. Haplotypes analysis reveals the genetic basis of type I CD36 deficiency. Sci Rep . 2024;14(1):23977. Andersen M, Lenhard B, Whatling C, et al. Alternative promoter usage of the membrane glycoprotein CD36. BMC Mol Biol . 2006;7:8. Hanawa H, Watanabe K, Nakamura T, et al. Identification of cryptic splice site, exon skipping, and novel point mutations in type I CD36 deficiency. J Med Genet . 2002;39(4):286-291. Zhang T, Li H, Jiang M, et al. Nanopore sequencing: flourishing in its teenage years. J Genet Genomics . 2024;51(12):1361-1374. Chen J, Liao G, He R, et al. Identification of a Novel HLA-G Allele, HLA-G*01:62, Using PolyseqOne and Oxford Nanopore Sequencing. HLA . 2025;106(1):e70295. Niculite CM, Enciu AM, Hinescu ME. CD 36: Focus on Epigenetic and Post-Transcriptional Regulation. Front Genet . 2019;10:680. Li L, Wu G, Liang L, et al. CD36 c.1328_1331dup: A variant causing platelet CD36 deficiency and its frequency in the Chinese population. Vox Sang . 2025. Epub ahead of print. Hendrickson JE, Tormey CA, Shaz BH. Red blood cell alloimmunization mitigation strategies. Transfus Med Rev . 2014;28(3):137-144. Wheeler MM, Lannert KW, Huston H, et al. Genomic characterization of the RH locus detects complex and novel structural variation in multi-ethnic cohorts. Genet Med . 2019;21(2):477-486. Marx V. Method of the year: long-read sequencing. Nat Methods . 2023;20(1):6-11. Oehler JB, Wright H, Stark Z, et al. The application of long-read sequencing in clinical settings. Hum Genomics . 2023;17(1):73. Lyu Q, Lin Y, Pan Y, et al. The polymorphism analysis for CD36 among platelet donors. Sci Rep . 2024;14(1):8534. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.docx SupplementaryFigure.docx Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 07 Jan, 2026 Reviews received at journal 28 Nov, 2025 Reviews received at journal 26 Nov, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers invited by journal 11 Nov, 2025 Editor assigned by journal 10 Nov, 2025 Editor invited by journal 10 Nov, 2025 Submission checks completed at journal 08 Nov, 2025 First submitted to journal 08 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8004674","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":546502159,"identity":"9d500f05-c359-4693-88af-1f5451ea5993","order_by":0,"name":"Shuang Liang","email":"","orcid":"","institution":"Shenzhen Blood Center","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Liang","suffix":""},{"id":546502160,"identity":"df4d286a-5702-45bb-9af4-d82ffc812efc","order_by":1,"name":"Tong Liu","email":"","orcid":"","institution":"Shenzhen Blood Center","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Liu","suffix":""},{"id":546502161,"identity":"c3b081a3-7410-4684-9d68-edad834154e8","order_by":2,"name":"Huatao Che","email":"","orcid":"","institution":"Shenzhen Blood Center","correspondingAuthor":false,"prefix":"","firstName":"Huatao","middleName":"","lastName":"Che","suffix":""},{"id":546502162,"identity":"2b5d4f3a-31bc-44e9-bf43-529a27dc6ba3","order_by":3,"name":"Wenxia Xia","email":"","orcid":"","institution":"Shenzhen Blood Center","correspondingAuthor":false,"prefix":"","firstName":"Wenxia","middleName":"","lastName":"Xia","suffix":""},{"id":546502163,"identity":"be0bae6c-f44c-45b9-b7a5-868d3661068c","order_by":4,"name":"Weiyi Fu","email":"","orcid":"","institution":"DAFEI biotechnology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Weiyi","middleName":"","lastName":"Fu","suffix":""},{"id":546502164,"identity":"db432f07-4d27-4ef4-800b-0ef7684d381a","order_by":5,"name":"Fan Wu","email":"","orcid":"","institution":"Shenzhen Blood Center","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Wu","suffix":""},{"id":546502165,"identity":"b9d45524-b3cf-4562-b83a-954e848888ac","order_by":6,"name":"Liyan Sun","email":"","orcid":"","institution":"Shenzhen Blood Center","correspondingAuthor":false,"prefix":"","firstName":"Liyan","middleName":"","lastName":"Sun","suffix":""},{"id":546502166,"identity":"8bbdd1c5-1ef7-4bee-87d8-2b7adcd80d6a","order_by":7,"name":"Yongshui Fu","email":"","orcid":"","institution":"Guangzhou First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongshui","middleName":"","lastName":"Fu","suffix":""},{"id":546502167,"identity":"f997bae7-e2dd-4656-836e-fed9a6f44c17","order_by":8,"name":"Faming Zhu","email":"","orcid":"","institution":"Blood Center of Zhejiang Province","correspondingAuthor":false,"prefix":"","firstName":"Faming","middleName":"","lastName":"Zhu","suffix":""},{"id":546502168,"identity":"083d1a15-7942-425c-b62f-b1ce6a0f19a0","order_by":9,"name":"Dawei Cui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIie3RMQuCQBTA8ReBLoeuJ9R3uBAicOir3BHoks0NDgeBq9/G1gvBlgtXR6e2qGhpik5bmg7dgu4Pj3vD+00HYDL9YkINBUBu98B4APH4INJGPksP4hzlrGmSYuJX4oxhGzBun4SWeHLtE1oWaC5EiEFGjKMN1RIi4hwzrsiBh3iUFoxjRPSkuuyfLfF3oMirD6njHFpCrJbwHsSrrw9MywhhCauFWvwUrfXEqUJ2fybB0s0kq29JMM1sqSdfIdp9ptX3XmWLAccmk8n0T70BamlG7XgYD3UAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Dawei","middleName":"","lastName":"Cui","suffix":""}],"badges":[],"createdAt":"2025-11-01 09:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8004674/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8004674/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-026-12701-2","type":"published","date":"2026-02-27T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96557006,"identity":"f93e92ae-08ee-49c8-b8b6-754254cc1fd1","added_by":"auto","created_at":"2025-11-23 11:51:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":905081,"visible":true,"origin":"","legend":"","description":"","filename":"Revised2FulllengthhaplotypereconstructionofCD36bylongreadsequencinguncoversanovelstructuralvariant.docx","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/2e9d8e1cfa6daf44b4387372.docx"},{"id":96557007,"identity":"41aebe73-45f0-430d-b541-5eb56dee516b","added_by":"auto","created_at":"2025-11-23 11:51:22","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11072,"visible":true,"origin":"","legend":"","description":"","filename":"284d789568c3431ea241acf575a92aa9.json","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/36d2b3b0e6b2f3bb1a287322.json"},{"id":96557014,"identity":"a532a0d1-d36c-4ec8-96f4-9539b409a6b5","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1798188,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/e7f3e383eb9c50e374c61885.docx"},{"id":96557023,"identity":"37b0314b-750e-431c-830c-51fe48d3f1d7","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114949,"visible":true,"origin":"","legend":"","description":"","filename":"284d789568c3431ea241acf575a92aa91enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/ab7b959ba3198455a48e98d9.xml"},{"id":96557057,"identity":"582dec11-c8d1-4282-803a-9d500726ac81","added_by":"auto","created_at":"2025-11-23 11:51:37","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":276343,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/5d9ac11e5a93080402121992.jpeg"},{"id":96557015,"identity":"82bb59c0-3193-4f89-89e1-c33e3e2af295","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":54351,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/95a25217dbec79fcd8df48fe.png"},{"id":96557022,"identity":"739e4ae3-91a6-492b-b384-6d682456c6ab","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":211282,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/b3bc7c63fe1598278026bbf8.jpeg"},{"id":96604875,"identity":"98169de6-0f7c-4231-a797-849d83286c7d","added_by":"auto","created_at":"2025-11-24 09:15:35","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74652,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/6c11277314c19ffdf61229c3.jpeg"},{"id":96557028,"identity":"fb0cf22a-66c2-4983-8e3b-2d36274cb243","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79446,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/75f8f0a2f85f42e96712fd5b.jpeg"},{"id":96557029,"identity":"c6918ca3-d9f3-4521-8d2e-1369586e8b61","added_by":"auto","created_at":"2025-11-23 11:51:24","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142089,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/0496217fa60001529a6e8677.jpeg"},{"id":96557025,"identity":"279d3af4-994e-412e-a7fb-441786e8bba4","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75923,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/d066426e6e3d374e960f5bb0.png"},{"id":96557018,"identity":"eb951492-9ef1-4fbe-9045-5a8c5a6eda58","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9204,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/902d3b7de8226b36c498d041.png"},{"id":96557021,"identity":"bca9af95-b181-4e18-96ca-cd98ace4b922","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127760,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/54943dc760999becd90e6754.png"},{"id":96557019,"identity":"936b9408-fbeb-49c0-9aa9-5085f1b32230","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61777,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/787d1965d9fa08ff39a1aa49.png"},{"id":96604918,"identity":"3d31addb-2519-4078-8a35-77b4f2b76c6f","added_by":"auto","created_at":"2025-11-24 09:16:03","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45548,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/fdce485537fe2e5dfca334b8.png"},{"id":96605690,"identity":"2992b764-5bb9-4563-9c5a-db6423afbe00","added_by":"auto","created_at":"2025-11-24 09:23:49","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":53768,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/b163c723898ade5f0108dd93.png"},{"id":96557024,"identity":"ba567f0f-a203-43dc-b1e6-9c966775f531","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111664,"visible":true,"origin":"","legend":"","description":"","filename":"284d789568c3431ea241acf575a92aa91structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/83c90267b2f189ba0dd99f1a.xml"},{"id":96557020,"identity":"d675e8ec-a473-480a-ad8a-20d7188df128","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120982,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/6b9c4a3d6e2aeb295abc4e9c.html"},{"id":96604701,"identity":"1048d1f8-cafd-445d-a911-c675f0985729","added_by":"auto","created_at":"2025-11-24 09:14:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":400512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic overview of identification for the full-length \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCD36\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/2a17ab609b77206e0ef0fb2d.png"},{"id":96557016,"identity":"42e4995c-4bd3-4e40-acf5-825696ee02f8","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted amplification and sequencing of the full-length CD36 gene.\u003c/strong\u003eRed and blue arrows depict alignment of haplotype sequences to the CD36 reference (GRCh38). White bars within arrows indicate base differences or insertions relative to the reference; SNP differences within overlapping amplicon regions enabled phasing of the four fragments into complete haplotypes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/1ed3000d88b3380248e4f0ff.png"},{"id":96557010,"identity":"02fb4683-b80e-4fe2-93db-54c9bae9c067","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":632144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the structural variant in the CD36 gene.\u003c/strong\u003e (A) Four deficient samples showed a consistent pattern: one haplotype amplified all four amplicons, while the other failed to amplify amplicons 2 and 3. A representative sample is shown. Red and blue arrows represent alignment to the CD36 reference sequence (GRCh38). Differences from the reference are indicated by white bars within arrows; blue/red lines/triangles denote insertions. (B) IGV visualization (PacBio WGS sample) showing markedly reduced read depth across the deletion region (black line). Reads aligned upstream of the breakpoint but failed downstream. Gray areas represent aligned regions; colored mismatches correspond to unaligned post-deletion sequences. (C) Assembled PacBio sequences from two samples. The blank region represents the deleted segment. Nucleotide sequences at the precise breakpoints are shown.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/5468d8007d56877e00a877d6.png"},{"id":96557012,"identity":"427cd478-c1dc-4322-b97d-cf43bca971a0","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":246850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGap-PCR screening for the structural variant in blood donors. \u003c/strong\u003e(A) Primer design schematic. CD36 5F/R primers flank the deletion breakpoints; CD36 6F/R primers (intron 7) amplify a 333-bp internal control fragment. (B) Agarose gel electrophoresis. Lane M: Marker; NC: Negative control; PC: Positive control; Arrows indicate three heterozygous carriers (Sample 69, 81 and 223) showing both 333-bp control and 832-bp deletion-specific bands.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/06b8fe727544cc538b29d0f0.png"},{"id":96604607,"identity":"836a01f8-d3ed-490c-94c7-6c89e4f7dfb4","added_by":"auto","created_at":"2025-11-24 09:14:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":241250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular and phenotypic characterization of heterozygous carriers of the structural variant.\u003c/strong\u003e (A) Nanopore sequencing confirmation of identical deletion breakpoints in three carriers. Haplotype sequences spanning the deletion junction (dashed box) show concordance with deficient cases. (B) Flow cytometric analysis of platelet CD36 surface expression. Histogram overlay demonstrates preserved expression in heterozygous carriers (blue: Sample 69; green: Sample 81; yellow: Sample 223) compared to positive (red, wild-type) and negative (purple, CD36-deficient) controls.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/bc58418514c5f7be4ea62adf.png"},{"id":103766631,"identity":"c1bb1863-0cd2-4068-a5c2-63461606db58","added_by":"auto","created_at":"2026-03-02 16:15:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2852732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/86ccca06-120f-4999-b29a-d25eea9ba56b.pdf"},{"id":96604876,"identity":"da476ab8-10c9-4204-9b3e-add3f413a5a6","added_by":"auto","created_at":"2025-11-24 09:15:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1798188,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/71dbffcc4c4f59df497e8c0e.docx"},{"id":96557017,"identity":"a9998cbd-1787-4369-814b-c993db4ac4e6","added_by":"auto","created_at":"2025-11-23 11:51:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":158943,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8004674/v1/3d1d695c50fb31c06e4b9fec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Full-length haplotype reconstruction of CD36 by long-read sequencing: uncovers a novel structural variant","fulltext":[{"header":"Key Points","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003eA long-read sequencing approach was established and successfully and firstly reconstructed the complete ~77kb CD36 haplotype.\u003c/li\u003e\n \u003cli\u003eCD36-deficient donors included a novel 19,971bp deletion-insertion structural variant without antigen dosage effect of heterozygotes.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eCD36, also known as glycoprotein IV (GPIV), is an 88-kDa transmembrane protein expressed on a wide range of cell types including platelets, monocytes, endothelial cells, and erythroid precursors, though expression on mature red blood cells (RBCs) is relatively low\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Initially identified as a platelet membrane component, CD36 has since been implicated in diverse physiological functions including lipid metabolism, innate immunity, and cell adhesion, and it also serves as a key antigen target in alloimmune thrombocytopenia disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCD36 deficiency is classified into two phenotypes: type I, characterized by complete absence of expression on both platelets and monocytes, and type II, defined by selective absence on platelets\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Although individuals with CD36 deficiency are often clinically asymptomatic, exposure to CD36-positive cells through blood transfusion or pregnancy can lead to alloimmunization and the production of anti-CD36 antibodies\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These antibodies are known to cause serious immune-mediated complications such as FNAIT, PTR, and posttransfusion purpura\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Importantly, CD36-negative phenotypes vary in prevalence across populations, reaching up to 11% in certain Asian cohorts, and have become increasingly relevant to transfusion practice in regions with active donor surveillance\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eCD36\u003c/em\u003e gene, located on chromosome 7q21.11, spans approximately 77 kb and includes 15 exons, 12 of which are coding\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Although over 30,000 variants are listed in the dbSNP database, only a small subset is known to affect protein expression\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Most studies have focused on coding variants and selected single nucleotide variants (SNVs), while the broader haplotype structure and upstream regulatory regions remain understudied\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Using PacBio sequencing, Xia et al. obtained\u0026thinsp;~\u0026thinsp;40 kb haplotypes from exon 2 to the 3\u0026rsquo; untranslated region (UTR) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, in some type I deficiency cases, causative variants could not be identified within these regions, suggesting that critical regulatory elements might reside in intron 1 or adjacent noncoding regions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we used four overlapping long-range polymerase chain reaction (PCR) amplicons (20\u0026thinsp;~\u0026thinsp;23 kb each) to generate full-length (~\u0026thinsp;77 kb) haplotype sequences of the \u003cem\u003eCD36\u003c/em\u003e gene using the Polyseq nanopore sequencing platform\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Applying this approach, we analyzed samples from 14 individuals with type I CD36 deficiency and 29 with type II deficiency. In three type I and one type II CD36 deficiency samples, we detected potential structural variants located in intron 1. Among them, two cases were further validated by PacBio whole-genome sequencing, which confirmed a 19,971 bp deletion (c.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA) with precise breakpoint resolution. Finally, we screened 300 routine blood donors using a PCR-based assay and estimated the allele frequency of this large deletion haplotype to be approximately 0.6%.\u003c/p\u003e"},{"header":"2. Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Sample Collection\u003c/h2\u003e\u003cp\u003eFourteen type I and twenty-nine type II CD36-deficient samples were randomly selected from the Shenzhen Blood Center CD36 Deficiency Bank. An additional 600 normal samples were collected from routine healthy donors at the same center. Peripheral blood (5.0 mL) was collected in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes for genomic DNA extraction and flow cytometry analysis. A graphical overview of this study is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All donors were of Han ethnicity and all samples were obtained with informed consent under ethical approval from the Shenzhen Blood Center Ethics Committee (SZBCMEC-2024-047).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Genomic DNA Extraction\u003c/h2\u003e\u003cp\u003eGenomic deoxyribonucleicacid (DNA) was isolated from EDTA-anticoagulated peripheral blood using the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics). DNA purity (A₂₆₀/A₂₈₀ ratio 1.8-2.0) and concentration were assessed by spectrophotometry (NanoDrop 8000), and extracts were normalized to 30 ng/\u0026micro;L in Tris-HCl/EDTA buffer for downstream use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 LR-PCR for Full-Length \u003cem\u003eCD36\u003c/em\u003e Gene\u003c/h2\u003e\u003cp\u003ePrimer sets (\u003cem\u003eCD36\u003c/em\u003e 1Forward/Reverse-4 Forward/Reverse) were designed to amplify regions spanning from the 5\u0026rsquo; UTR to the 3\u0026rsquo; UTR of the \u003cem\u003eCD36\u003c/em\u003e gene, covering a total length of approximately 77 kb and including selected intronic sequences. Four sets were developed to generate amplicons ranging from 21\u0026ndash;23 kb in length, with \u0026ge;\u0026thinsp;3 kb overlap between adjacent fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Long-range PCR (LR-PCR) was performed using ApexHF HS DNA Polymerase CL (Accurate Biology, AG12204) according to the manufacturer\u0026rsquo;s instructions. PCR cycling consisted of a three-step program: 98\u0026deg;C for 10 s, 65\u0026deg;C for 10 s, and 72\u0026deg;C for 3.5 min, for 30 cycles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer pairs used for full-length CD36 gene amplification and gap-PCR assay.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence (5\u0026rsquo;\u0026rarr;3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSize\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCoverage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUsage\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 1-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ectccctcaccacctatccctataagct\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e20.6 kb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e5\u0026rsquo;UTR\u0026thinsp;~\u0026thinsp;exon 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eLR-PCR primers for LRS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 1-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ectgccactgcttcatcaaccaacttta\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 2-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecaaagtggctacggcgattgtaaggatc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e22 kb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eintron 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 2-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eacagtagtgtcacctcccgtcatctgg\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 3-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003etccaaattcagactcggattccattcat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e20.6 kb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eexon 2\u0026thinsp;~\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 3-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eaccttaccatacacttgcagcagaaat\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 4-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eggcttagacagtaaatgctatgaaccaag\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e23 kb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eexon 4\u0026thinsp;~\u0026thinsp;14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 4-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecaccgtcttacactttgctacttcttaca\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 5-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGTGTCAAACTGTCATCGGGGTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e20 kb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eexon 2\u0026thinsp;~\u0026thinsp;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003edeletion-flanking primers for gap-PCR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 5-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGACCCTTTTCCACAGTGCTTTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 6-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAGAGAGATGTGGAGGGGAGTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e290 bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eintron 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003econtrol primers for gap-PCR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD36 6-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCTTGCCCCTTCCTCCCTTTA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eNote\u003c/b\u003e: F: forward, R: reverse.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Library Preparation, Nanopore Sequencing and Bioinformatic Analysis\u003c/h2\u003e\u003cp\u003eLibrary preparation was performed using the commercial PY-DTB101/102 and PY-BLP101 kits (Polyseq Biotech Co. Ltd., China) according to the manufacturer\u0026rsquo;s instructions. Prepared libraries were loaded onto PY-NFC001 flow cells and sequenced for 12 hours on the PolyseqOne nanopore sequencing platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.polyseq.com\u003c/span\u003e\u003c/span\u003e). Basecalling and demultiplexing were performed using Kant v1.0.1 in high-accuracy mode (processing rate: 420 bases per second). Raw reads underwent demultiplexing with automated barcode identification. For CD36 sequence analysis, reads were first aligned to the T2T-CHM13v2.0 reference genome and error-corrected using NanoFix-AI (DAFEI Biotechnology, Guangzhou, China) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. FASTQ reads corresponding to individual CD36 alleles were extracted using bedtools (v2.30.0). A custom Python workflow was then employed to convert these reads into clustered BAM files. Finally, the clustered reads were re-aligned to the CD36 reference gene sequence using pbmm2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Whole-Genome Sequencing Analysis\u003c/h2\u003e\u003cp\u003eLong-read whole-genome sequencing was performed on the PacBio Revio platform (Pacific Biosciences), generating 31.2 Gb and 52.4 Gb of HiFi reads with median qualities of Q35. Coverage depth across the \u003cem\u003eCD36\u003c/em\u003e gene was 25\u0026times; and 41\u0026times;, respectively. Reads were aligned to the human reference genome (CHM13v2.0) using minimap2 (v2.14-r883) with default parameters. Large deletions were detected using software developed by DAFEI Biotechnology (Guangzhou, China). Coverage was assessed with mosdepth, and deletion breakpoints/junctions were manually verified using the Integrative Genomics Viewer (IGV). \u003cem\u003eCD36\u003c/em\u003e sequence data were visualized in SnapGene for clear presentation of structural findings.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Population Screening for 19,971-bp Deletion-Insertion Structural Variant\u003c/h2\u003e\u003cp\u003eWe developed a multiplex gap-PCR assay to screen 600 blood donors for the recurrent 19,971bp deletion-insertion structural variant spanning exons 2\u0026ndash;3. Two primer pairs were designed based on the CD36 gene sequence (GenBank ID: NG_008192.1): control primers (\u003cem\u003eCD36\u003c/em\u003e 6F/R) amplifying a 333-bp fragment in intron 7, and deletion-flanking primers (\u003cem\u003eCD36\u003c/em\u003e 5F/R) positioned 571 bp upstream (5F) and 261 bp downstream (5R) of the deletion boundaries. All primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. PCR amplification was performed in multiplex format under the following conditions: 30 cycles of 98\u0026deg;C for 10 s, 63\u0026deg;C for 30 s, and 72\u0026deg;C for 1 min, with pre-validated positive and negative controls included in each run. All candidate positives underwent confirmation through nanopore sequencing (Polyseq platform) to verify breakpoint identity with whole-genome sequencing data and determine zygosity status.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Flow cytometric analysis for CD36 Expression on Platelets\u003c/h2\u003e\u003cp\u003eCD36 surface expression on platelets was assessed by flow cytometry using fresh whole blood samples processed within 24 hours of collection. Platelet-rich plasma (PRP) was isolated from EDTA-anticoagulated blood by centrifugation at 200 \u0026times;g for 10 min at room temperature. Following three washes with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 1 mM EDTA (PBS-BSA-EDTA), platelets were resuspended at 1\u0026times;10⁷ cells/ml. For staining, 20 \u0026micro;L platelet suspension was incubated with 2 \u0026micro;L FITC-conjugated anti-CD36 antibody (clone FA6-152; BD Biosciences, USA) for 20 min at room temperature in the dark. After three washes with PBS-BSA-EDTA, cells were resuspended in 500 \u0026micro;L PBS. Analysis was performed on a FACSCanto II flow cytometer (BD Biosciences) with platelets gated by characteristic forward/side scatter (FSC/SSC) profiles. CD36 expression was quantified as mean fluorescence intensity (MFI) using FACSDiva software v8.0.1 (BD Biosciences).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Reconstruction of Full-Length \u003cem\u003eCD36\u003c/em\u003e Haplotypes by Nanopore Sequencing\u003c/h2\u003e\u003cp\u003eFull-length (~\u0026thinsp;77 kb) \u003cem\u003eCD36\u003c/em\u003e haplotypes were reconstructed by SNP-based phasing of four overlapping 21\u0026ndash;23 kb amplicons, sequenced using the Polyseq nanopore platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Using this approach, we analyzed samples from 14 type I and 29 type II CD36-deficient individuals. All identified exonic variants (including those within 10 bp of exon-intron boundaries) present on each haplotype are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This represents the first successful reconstruction of the complete\u0026thinsp;~\u0026thinsp;77 kb \u003cem\u003eCD36\u003c/em\u003e haplotype, providing a comprehensive view of its genetic architecture.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenetic variations in CD36 exons among haplotype sequences from CD36 deficient samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHaplotype 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHaplotype 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.430-1G\u0026thinsp;\u0026gt;\u0026thinsp;C; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1229T\u0026thinsp;\u0026gt;\u0026thinsp;C*; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.380C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.430-2A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.609\u0026thinsp;+\u0026thinsp;7A\u0026thinsp;\u0026gt;\u0026thinsp;G*; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.1409C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1006\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.1409C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI-14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1163A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.220C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.275C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.332_333delCA; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.429\u0026thinsp;+\u0026thinsp;3insG*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.847G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.1409C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.609\u0026thinsp;+\u0026thinsp;7A\u0026thinsp;\u0026gt;\u0026thinsp;G*; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1228_1239delATTGTGCCTATT; c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1340_1343dupTCTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.681C\u0026thinsp;\u0026gt;\u0026thinsp;A*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eII-29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Represents the new variants identified in this study.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Variant Spectrum and Structural Variants in Type I and II CD36 Deficiency\u003c/h2\u003e\u003cp\u003eAnalysis of the haplotypes derived from 14 type I deficiency samples revealed that all harbored either previously reported pathogenic point variations or complex structural variants, including 13 compound heterozygous variants and 1 homozygous variant. The most frequent variant was c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C and c.1227delTATTGTGCCTAT, both detected in 32.1% (9/28) of haplotypes. The remaining 35.7% (10/28) of haplotypes harbored a spectrum of other variants, including the novel 19,971-bp deletion-insertion structural variant (10.7%, 3/28) and multiple other point variations and indels as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In contrast, among the 28 type II deficiency samples, 22 exhibited variants in the \u003cem\u003eCD36\u003c/em\u003e gene (18 compound heterozygous variants and 4 heterozygous variants), the predominant variant was c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C, identified in 20.7% (12/58; assuming a comparable denominator context, or specify total type II haplotypes if known). Thus, we successfully obtained complete haplotype sequences in 43 CD36 deficiency samples using the Polyseq nanopore platform, enabling phased variant calling that was not achievable by previous methods. All haplotype sequences have been submitted to the NCBI database (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenetic variations of the CD36 gene in CD36 deficient samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGenetic variations\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eExon/Intron\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eType I deficiency\u003c/p\u003e\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;28)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eType II deficiency\u003c/p\u003e\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;58)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAccession no.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFrequency\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFrequency\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.220C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV866985\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.275C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV866988\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.332_333delCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV808479\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.380C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV808480\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.429\u0026thinsp;+\u0026thinsp;3insG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eintron5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV866987\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.430-1G\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eintron5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV866984\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.430-2A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eintron5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV832518\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.609\u0026thinsp;+\u0026thinsp;7A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eintron6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV808482\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.681C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV855215\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.847G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV866986\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1006\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eintron10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV866989\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV808481\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1156C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.1409C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 12, 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV855214\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1163A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV855217\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1227delTATTGTGCCTAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e30.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV786778\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1229T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.33%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV855218\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1340_1343dupTCTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV855216\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ec.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eexon 2\u0026ndash;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.00%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.67%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePV866990\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 A Novel Structural Variant Spanning \u003cem\u003eCD36\u003c/em\u003e Exons 2\u0026ndash;3\u003c/h2\u003e\u003cp\u003eIn four CD36-deficient samples (3 type I, 1 type II), nanopore sequencing revealed consistent amplification failure of amplicons 2\u0026ndash;3 in one haplotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), suggesting structural variants. To further characterize this inspection, we performed whole-genome sequencing on two deficient samples (I-13 and II-22) using the PacBio Revio platform. This generated 31.2 Gb and 52.4 Gb of HiFi reads, respectively, with a median quality score of Q35; the majority of reads exceeded 10 kb in length (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Visualization using IGV revealed a sharp drop in coverage and disrupted alignment across the second half of intron 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Subsequent de novo assembly identified a 19,971 bp deletion-insertion structural variant (c.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA, NG_008192.1: g.33588_g.53560delinsCCAATGCTAAGGTTGA) in one haplotype of each sample. This deletion spans positions c.1-15966 (following a 16-bp insertion) to c.120\u0026thinsp;+\u0026thinsp;3887 (relative to the \u003cem\u003eCD36\u003c/em\u003e reference sequence, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), thereby removing exons 2 and 3, including the translation initiation site located in exon 3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Population Screening for 19,971-bp Deletion-Insertion Structural Variant\u003c/h2\u003e\u003cp\u003eUnder the established gap-PCR conditions, amplification of the wild-type allele (20.8-kb) consistently failed, whereas the deletion allele yielded a distinct 832-bp fragment. Consequently, samples exhibiting both the 333-bp control and 832-bp bands were classified as potential carriers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Screening of 600 blood donors using this assay identified three heterozygous carriers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating a population carrier frequency of 0.50% (3/600), and the full uncropped Gels and Blots image(s) were displayed as \u0026ldquo;Supplementary file\u0026rdquo;. Nanopore sequencing confirmed that the breakpoints in these carriers were identical to those observed in the CD36-deficient cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Critically, flow cytometric analysis demonstrated preserved CD36 antigen expression on platelets in all three heterozygotes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that the deletion alone does not cause haploinsufficiency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCD36 deficiency poses a significant risk in transfusion medicine due to its potential to induce alloantibodies that can cause PTR and FNAIT, particularly in Asian and African populations where its prevalence is higher\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Despite its clinical importance, the genetic architecture of CD36 deficiency remains incompletely understood. Although multiple point variations and small indels in exonic and splice-site regions have been reported, these do not fully explain the observed phenotypic variation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Evidence suggests that structural variants and regulatory elements in noncoding regions may contribute to the pathogenesis, especially in type I deficiency\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, the highly polymorphic nature of the \u003cem\u003eCD36\u003c/em\u003e locus complicates the establishment of clear genotype-phenotype correlations\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we applied long-read sequencing to reconstruct full-length \u003cem\u003eCD36\u003c/em\u003e haplotypes, achieving comprehensive resolution of the ~\u0026thinsp;77 kb locus for the first time. By integrating the previously reported c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C promoter variant and c.1228-1239delATTGTGCCTATT deletion variant\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, as well as the novel 19,971-bp deletion-insertion structural variant identified here, we successfully explained the molecular basis of all 28 type I deficiency haplotypes. Compared with variants were detected in all haplotypes of type I deficiency samples, no CD36 gene variants were detected in either haplotype in 7 type II deficiency samples. Furthermore, although I-02 and II-18 had the same allele genotype (c.1228_1239delATTGTGCCTATT on one allele and c.-132A\u0026thinsp;\u0026gt;\u0026thinsp;C on the other), their CD36 antigen expression levels exhibit significant differences. All of the above suggesting that other mechanisms-such as regulatory element alterations, aberrant splicing, or epigenetic changes-may underlie this phenotype\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Our amplicon-based sequencing strategy provides a foundation for further investigation into these unresolved cases.\u003c/p\u003e\u003cp\u003eThe discovery of the 19,971-bp deletion within \u003cem\u003eCD36\u003c/em\u003e carries critical implications for molecular diagnostics. Our identification of the structural variant, which removes the translation initiation site in exon 3, explains its severe impact on protein expression when homozygous or compound heterozygous. Importantly, this structural variant was undetectable by Sanger sequencing in all four cases where it occurred, as conventional methods failed to amplify across the breakpoints localized deep within introns 1 and 3. This diagnostic gap likely led to prior misclassification of compound heterozygotes as homozygotes for point variations (e.g., in exons 4\u0026ndash;14), underscoring a fundamental limitation of targeted exon sequencing.\u003c/p\u003e\u003cp\u003ePopulation screening revealed a carrier frequency of 0.50% for this deletion. Heterozygous individuals exhibited normal CD36 expression, indicating that the variant does not cause haploinsufficiency. However, compound heterozygosity, where this structural variant coexists with another pathogenic variant on the alternate allele, can manifest the CD36-deficient phenotype. The asymptomatic carriage of this structural variant in the general population carries important practical implications: implementing targeted screening for CD36-negative platelet donors in regions with high allele prevalence may provide an effective preventive strategy to mitigate alloimmunization risks\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMoreover, our long-read sequencing approach overcomes critical limitations of conventional genotyping methods\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Previous Sanger sequencing of exonic regions, while capable of identifying point variations and small indels, could not resolve the phased haplotype structure of \u003cem\u003eCD36\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e. This frequently led to diagnostic ambiguities, such as the misinterpretation of hemizygous large deletions as homozygous point variations due to amplification failure of the deleted allele. Our method enables unambiguous phasing and accurate detection of structural variants, offering a more reliable molecular diagnostic framework for CD36 deficiency. In summary, this study resolves a major diagnostic gap in \u003cem\u003eCD36\u003c/em\u003e genotyping by demonstrating the utility of long-read sequencing for comprehensive haplotype analysis. Our findings support the integration of population screening and advanced genomic technologies into clinical practice to better manage transfusion-related risks associated with CD36 deficiency.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study establishes the first full-length haplotype map of the \u003cem\u003eCD36\u003c/em\u003e gene (~\u0026thinsp;77 kb) through long-read sequencing of four overlapping amplicons. This approach enabled the discovery of a novel recurrent 19,971-bp deletion-insertion structural variant (c.1-15966_c.120\u0026thinsp;+\u0026thinsp;3887delinsCCAATGCTAAGGTTGA) spanning intron 1 to intron 3, which eliminates exons 2\u0026ndash;3 and the translation initiation site. The deletion was identified in three type I and one type II deficiency haplotypes. Population screening of blood donors revealed a 0.50% allele frequency, with flow cytometry confirming normal CD36 expression on platelets in all heterozygous carriers. These findings resolve a critical diagnostic blind spot for structural variants undetectable by conventional methods and provide essential insights for clinical management of CD36-related alloimmune risks in transfusion medicine.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFNAIT - Fetal/Neonatal Alloimmune Thrombocytopenia\u003c/p\u003e\n\u003cp\u003ePTR -\u0026nbsp;Platelet Transfusion Refractoriness\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGPIV - Glycoprotein IV\u003c/p\u003e\n\u003cp\u003eRBCs - Red Blood Cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSNV \u0026ndash; Single Nucleotide Variant\u003c/p\u003e\n\u003cp\u003eUTRs \u0026ndash;Untranslated Regions\u003c/p\u003e\n\u003cp\u003ePCR - Polymerase Chain Reaction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDNA - Deoxyribonucleicacid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEDTA - Ethylenediaminetetraacetic Acid\u003c/p\u003e\n\u003cp\u003eLR-PCR - Long-range PCR\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePRP - Platelet-Rich Plasma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBS - Phosphate Buffered Saline\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBSA - Bovine Serum Albumin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMFI - Fluorescence Intensity\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eDeclaration of competing interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics Approval and Consent\u003c/p\u003e\n\u003cp\u003eThis project was conducted in accordance with the guidelines of the Helsinki Declaration, and approved by the Shenzhen Blood Center Ethics Committee (SZBCMEC-2024-047). All involved participants provided written informed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for Publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0007933 (https://db.cngb.org/data_resources/project/CNP0007933/). The original data and materials from this research are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank the blood donors and patients in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Shenzhen Municipality (No. JCYJ20230807154000002), Guangdong Medical Foundation (No. B2025582), Shenzhen Key Medical Discipline Construction Fund (No. SZXK070) and Sanming Project of Medicine in Shenzen Municipality (No. SZSM202311032), Key Research and Development Plan of Department of Science and Technology of Zhejiang Province (No. 2024C03156).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; Contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShuang Liang:\u0026nbsp;\u003c/strong\u003eOriginal draft, Investigation, Funding acquisition. \u003cstrong\u003eTong Liu:\u0026nbsp;\u003c/strong\u003eMethodology, Data curation. \u003cstrong\u003eHuatao Che:\u0026nbsp;\u003c/strong\u003eMethodology, Formal analysis. \u003cstrong\u003eWenxia Xia:\u0026nbsp;\u003c/strong\u003eMethodology, Data curation. \u003cstrong\u003eWeiyi Fu:\u0026nbsp;\u003c/strong\u003eData acquisition. \u003cstrong\u003eFan Wu:\u0026nbsp;\u003c/strong\u003eMethodology. \u003cstrong\u003eLiyan Sun:\u0026nbsp;\u003c/strong\u003eMethodology, Statistical analyses. \u003cstrong\u003eYongshui Fu:\u0026nbsp;\u003c/strong\u003eWriting-review ＆ editing. \u003cstrong\u003eFaming Zhu:\u0026nbsp;\u003c/strong\u003eWriting-review ＆ editing.\u003cstrong\u003e\u0026nbsp;Dawei Cui:\u0026nbsp;\u003c/strong\u003eWriting-review ＆ editing, Supervision, Funding acquisition. All the authors have read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003evan Schravendijk MR, Handunnetti SM, Barnwell JW, et al. Normal human erythrocytes express CD36, an adhesion molecule of monocytes, platelets, and endothelial cells. \u003cem\u003eBlood\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e1992;80(8):2105-2114.\u003c/li\u003e\n\u003cli\u003eTian J, Liu W, Gao W, et al. Molecular cloning and gene/protein expression of FAT/CD36 from grass carp (Ctenopharyngodon idella) and the regulation of its expression by dietary energy. \u003cem\u003eFish Physiol Biochem\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2017;43(3):875-888.\u003c/li\u003e\n\u003cli\u003eChen Y, Zhang J, Cui W, et al. CD36, a signaling receptor and fatty acid transporter that regulates immune cell metabolism and fate. \u003cem\u003eJ Exp Med.\u003c/em\u003e 2022;219(6):e20211314.\u003c/li\u003e\n\u003cli\u003eTzeng HT, Chyuan IT, Chen WY. Shaping of Innate Immune Response by Fatty Acid Metabolite Palmitate. \u003cem\u003eCells\u003c/em\u003e. 2019. 8(12):1633.\u003c/li\u003e\n\u003cli\u003eCaruso JA, Wang X, Murrow LM, et al. Loss of PPAR\u0026gamma; activity characterizes early protumorigenic stromal reprogramming and dictates the therapeutic window of opportunity. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2023;120(42):e2303774120.\u003c/li\u003e\n\u003cli\u003eMasuda Y, Tamura S, Matsuno K, et al. Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects. \u003cem\u003eThromb Res.\u003c/em\u003e 2015;135(5):951-957.\u003c/li\u003e\n\u003cli\u003eOno R, Miyauchi H, Kobayashi Y. CD36 deficiency. \u003cem\u003eQJM: An International Journal of Medicine\u003c/em\u003e. 2025;118(5):363-364.\u003c/li\u003e\n\u003cli\u003eWu Y, Chen D, Xu X, et al. Hydrops fetalis associated with anti‐CD36 antibodies in fetal and neonatal alloimmune thrombocytopenia: possible underlying mechanism. \u003cem\u003eTransfus Med\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2020;30(5):361-368.\u003c/li\u003e\n\u003cli\u003eSchmidt AE, Sahai T, Refaai MA, et al. Severe Platelet Transfusion Refractoriness in Association with Antibodies Against CD36. \u003cem\u003eLab Med\u003c/em\u003e. 2020;51(5):540-544.\u003c/li\u003e\n\u003cli\u003eKhatri SS, Curtis BR, Yamada C. A case of platelet transfusion refractoriness due to anti-CD36 with a successful treatment outcome. \u003cem\u003eImmunohematology\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2019;35(4):139-144.\u003c/li\u003e\n\u003cli\u003eXu X, Li L, Xia W, et al. Successful management of a hydropic fetus with severe anemia and thrombocytopenia caused by anti-CD36 antibody. \u003cem\u003eInt J Hematol\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2018;107(2):251-256.\u003c/li\u003e\n\u003cli\u003eChen D, Liang H, Xu X, et al. Inhibition of terminal complement complex formation alleviates murine antibody-mediated TRALI. \u003cem\u003eBlood\u003c/em\u003e. 2025;146(6):759-764.\u003c/li\u003e\n\u003cli\u003eMasuda Y, Tamura S, Matsuno K, et al. Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects. \u003cem\u003eThromb Res\u003c/em\u003e. 2015;135(5):951-957.\u003c/li\u003e\n\u003cli\u003eKashiwagi H, Tomiyama Y, Kosugi S, et al. Identification of molecular defects in a subject with type I CD36 deficiency. \u003cem\u003eBlood\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e1994;83(12):3545-3552.\u003c/li\u003e\n\u003cli\u003eRać ME, Safranow K, Poncyljusz W. Molecular basis of human CD36 gene mutations. \u003cem\u003eMol Med\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2007;13(5-6):288-296.\u003c/li\u003e\n\u003cli\u003eMasuda Y, Tamura S, Matsuno K, et al. Diverse CD36 expression among Japanese population: defective CD36 mutations cause platelet and monocyte CD36 reductions in not only deficient but also normal phenotype subjects. \u003cem\u003eThromb Res\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2015;135(5):951-957.\u003c/li\u003e\n\u003cli\u003eXu X, Liu Y, Hong X, et al. Variants of CD36 gene and their association with CD36 protein expression in platelets. \u003cem\u003eBlood Transfus\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2014;12(4):557-564.\u003c/li\u003e\n\u003cli\u003eMadan N, Ghazi AR, Kong X, et al. Functionalization of CD36 cardiovascular disease and expression associated variants by interdisciplinary high throughput analysis. \u003cem\u003ePLoS Genet\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2019;15(7):e1008287.\u003c/li\u003e\n\u003cli\u003eXia W, Chen D, Li X, et al. Haplotypes analysis reveals the genetic basis of type I CD36 deficiency. \u003cem\u003eSci Rep\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e2024;14(1):23977.\u003c/li\u003e\n\u003cli\u003eAndersen M, Lenhard B, Whatling C, et al. Alternative promoter usage of the membrane glycoprotein CD36. \u003cem\u003eBMC Mol Biol\u003c/em\u003e. 2006;7:8.\u003c/li\u003e\n\u003cli\u003eHanawa H, Watanabe K, Nakamura T, et al. Identification of cryptic splice site, exon skipping, and novel point mutations in type I CD36 deficiency. \u003cem\u003eJ Med Genet\u003c/em\u003e. 2002;39(4):286-291.\u003c/li\u003e\n\u003cli\u003eZhang T, Li H, Jiang M, et al. Nanopore sequencing: flourishing in its teenage years.\u003cem\u003e J Genet Genomics\u003c/em\u003e. 2024;51(12):1361-1374.\u003c/li\u003e\n\u003cli\u003eChen J, Liao G, He R, et al. Identification of a Novel HLA-G Allele, HLA-G*01:62, Using PolyseqOne and Oxford Nanopore Sequencing. \u003cem\u003eHLA\u003c/em\u003e. 2025;106(1):e70295.\u003c/li\u003e\n\u003cli\u003eNiculite CM, Enciu AM, Hinescu ME. CD 36: Focus on Epigenetic and Post-Transcriptional Regulation. \u003cem\u003eFront Genet\u003c/em\u003e. 2019;10:680.\u003c/li\u003e\n\u003cli\u003eLi L, Wu G, Liang L, et al. CD36 c.1328_1331dup: A variant causing platelet CD36 deficiency and its frequency in the Chinese population. \u003cem\u003eVox Sang\u003c/em\u003e. 2025. Epub ahead of print.\u003c/li\u003e\n\u003cli\u003eHendrickson JE, Tormey CA, Shaz BH. Red blood cell alloimmunization mitigation strategies. \u003cem\u003eTransfus Med Rev\u003c/em\u003e. 2014;28(3):137-144.\u003c/li\u003e\n\u003cli\u003eWheeler MM, Lannert KW, Huston H, et al. Genomic characterization of the RH locus detects complex and novel structural variation in multi-ethnic cohorts. \u003cem\u003eGenet Med\u003c/em\u003e. 2019;21(2):477-486.\u003c/li\u003e\n\u003cli\u003eMarx V. Method of the year: long-read sequencing. \u003cem\u003eNat Methods\u003c/em\u003e. 2023;20(1):6-11.\u003c/li\u003e\n\u003cli\u003eOehler JB, Wright H, Stark Z, et al. The application of long-read sequencing in clinical settings. \u003cem\u003eHum Genomics\u003c/em\u003e. 2023;17(1):73.\u003c/li\u003e\n\u003cli\u003eLyu Q, Lin Y, Pan Y, et al. The polymorphism analysis for CD36 among platelet donors. \u003cem\u003eSci Rep\u003c/em\u003e. 2024;14(1):8534.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CD36, Full-length haplotype, Long-Read Sequencing, Structural variant","lastPublishedDoi":"10.21203/rs.3.rs-8004674/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8004674/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003eCD36 deficiency predisposes to fetal/neonatal alloimmune thrombocytopenia (FNAIT) and platelet transfusion refractoriness (PTR), yet its genetic architecture remains incompletely understood. This study employed long-read sequencing to reconstruct full-length \u003cem\u003eCD36 \u003c/em\u003ehaplotypes, aiming to identify its genetic backgrounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe developed a long-read sequencing approach to reconstruct full-length \u003cem\u003eCD36 \u003c/em\u003ehaplotypes from four overlapping amplicons. Samples from 43 CD36-deficient individuals (14 type I, 29 type II) were analyzed. Structural variants were validated via PacBio whole-genome sequencing, platelet CD36 expression was quantified by flow cytometry, and a gap-PCR assay screened 600 blood donors for large deletions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eFull-length 77-kb \u003cem\u003eCD36 \u003c/em\u003ehaplotypes were reconstructed for the first time using overlapping amplicons. All 28 haplotypes from type I deficiency samples harbored known or novel variations, including a novel structural variant (c.1-15966_c.120 + 3887delinsCCAATGCTAAGGTTGA, 19,971 bp deletion-insertion) spanning intron 1-3 that eliminates exons 2/3 and the translation initiation site. Among 58 haplotypes from type II cases, pathogenic variants were identified in only 26/58 (44.8%) (including one haplotype carrying the novel structural variant), while 32/58 (55.2%) lacked detectable variations. Gap-PCR screening revealed a 0.50% carrier frequency for this structural variant in blood donors; all heterozygotes exhibited wild-type \u003cem\u003eCD36 \u003c/em\u003eon the alternate allele with normal platelet CD36 expression confirmed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eA long-read sequencing approach with four overlapping amplicons was developed to successfully reconstruct the full-length \u003cem\u003eCD36 \u003c/em\u003ehaplotypes, which overcamelimitations of conventional genotyping and provided a robust technical foundation for identifying structural variants.\u003c/p\u003e","manuscriptTitle":"Full-length haplotype reconstruction of CD36 by long-read sequencing: uncovers a novel structural variant","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-23 11:51:18","doi":"10.21203/rs.3.rs-8004674/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-07T11:49:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T19:35:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T20:56:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104968301782568770230044214854089714928","date":"2025-11-17T18:48:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195443820985321311147440446606742362486","date":"2025-11-17T07:15:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214282118157734830688765141025514857443","date":"2025-11-12T04:27:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T03:43:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-10T19:51:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-10T19:39:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-08T15:11:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-11-08T15:08:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"467f0512-2f4c-44a9-b2fc-5a4c0b4f1091","owner":[],"postedDate":"November 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:13:27+00:00","versionOfRecord":{"articleIdentity":"rs-8004674","link":"https://doi.org/10.1186/s12864-026-12701-2","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2026-02-27 15:57:50","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-11-23 11:51:18","video":"","vorDoi":"10.1186/s12864-026-12701-2","vorDoiUrl":"https://doi.org/10.1186/s12864-026-12701-2","workflowStages":[]},"version":"v1","identity":"rs-8004674","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8004674","identity":"rs-8004674","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}