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. In particular, the contribution of large structural variants has been difficult to assess using conventional genotyping approaches. This study aimed to reconstruct full-length CD36 haplotypes using long-read sequencing and to systematically characterize its genetic architecture.

METHODS

We developed a long-read sequencing approach to reconstruct full-length (~ 77 kb) 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 CD36 haplotypes were reconstructed for the first time using overlapping amplicons. Candidate pathogenic variants were identified in all 28 haplotypes from type I deficiency samples, including a novel structural variant (c.1-15966_c.120 + 3887 delinsCCAATGCTAAGGTTGA, 19,971 bp deletion-insertion) spanning intron 1-3 that eliminates exons 2/3 and the canonical translation initiation site. Among 58 haplotypes from type II cases, potentially 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 heterozygous carriers showed normal platelet CD36 expression, indicating absence of haploinsufficiency.

CONCLUSIONS

This study establishes a robust PolySeq nanopore sequencing-based framework for full-length CD36 haplotype reconstruction and identifies a large structural deletion as a major, previously underrecognized candidate genetic mechanism underlying type I CD36 deficiency. Our findings suggest that many unresolved cases of type I deficiency may be attributable to cryptic structural variants missed by conventional methods, highlighting the necessity of long-read sequencing for accurate molecular diagnosis.