A Novel CTLA-4 Deletion variant in a Child with Refractory Autoimmune Hemolytic Anemia: Molecular and Functional Characterization

Feng Chen¹Siyu Lei²Caihui Yuan¹Zhongjin Xu¹Qian Wan¹Chongjun Wu^1*Ting Xiong^1*

• ¹Jiangxi Provincial Children's Hospital, Nanchang, China
• ²Nanchang University Jiangxi Medical College, Nanchang, China

Abstract: Objective: As a critical immune checkpoint, cytotoxic T-lymphocyte-associated protein 4(CTLA-4)deficiency is a well-established cause of inborn errors of immunity. This study characterizes a novel CTLA-4 deletion variant identified in a pediatric case of refractory autoimmune hemolytic anemia (AIHA), with the aim of delineating the clinical profile and elucidating the underlying pathogenic mechanism. Methods: Trio-based whole-exome sequencing (WES) was performed on peripheral blood samples from a 6-year-old female with refractory AIHA and her parents. Candidate variants were validated by Sanger sequencing. Structural modeling of mutant CTLA-4 was conducted, followed by in vitro functional assays in 293T cells to assess mRNA transcription (qPCR) and protein expression (Western blot). Results: A CTLA-4 (c.362_391del) variant was identified within the immunoglobulin V-set domain of the CTLA-4 protein. In vitro experiments demonstrated significant reductions in both mRNA and protein expression levels caused by this variant. Conclusion: The CTLA-4 (c.362_391del) variant may contribute to refractory AIHA in children. This case highlights the potential necessity of including CTLA-4 variants in the differential diagnosis of pediatric AIHA, particularly when conventional therapies prove ineffective, and warrants further validation in larger cohorts.