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

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.