Molecular basis of anti-CD36 alloimmunization: transcriptomic evidence linking intronic splice-site mutations to CD36...

CD36 deficiency is a genetic disorder that can lead to fetal/neonatal alloimmune thrombocytopenia or platelet transfusion refractoriness. While CD36 deficiency caused by exonic mutations are well established, the RNA splicing mechanism by intronic mutations remains poorly defined.

CD36 deficiency patients were analyzed by long-read sequencing and haplotype analysis. RNA sequencing and allele-specific analysis validated mutation-splicing associations in patients. CD36 aberrant transcript plasmids were constructed and assessed by flow cytometry. Exome sequencing of healthy donors determined mutation frequencies.

We found that population screening revealed a carrier frequency of approximately 2/800 for c.430-1G>C mutation and ~3/800 for c.429 + 3insG mutation among healthy Chinese donors. This study includes seven type I CD36 deficiency patients with PTR or FNAIT. Allele-specific analysis confirmed mutant allele enrichment in aberrant transcripts (P = 0.024). Long-read sequencing showed that one patient only had two intronic mutations (c.430-1G>C and c.429 + 3insG). Indicating intronic mutation was significantly linked to type I CD36 deficiency. Specifically, c.430-1G>C mutation induced aberrant exon 6 splicing, resulting in 17-bp pseudo-intron insertion or complete exon 6 skipping (28.6% or 30.3% skipped); the c.429 + 3insG mutation caused exon 5 skipping (38.3% skipped). Functional assays showed that the c.430-1G>C mutation led to complete loss of CD36 protein expression.

This study establishes that intronic mutations alone can also induce CD36 deficiency by impairing mRNA splicing, thereby extending the known mutational landscape of CD36 deficiency and reinforcing the necessity of screening intronic mutations in diagnostic and pathogenetic investigations.