Whole PROC Gene Sequencing to Explain Genetically Unresolved Protein C Deficiencies

Abstract

Background

With exon-focused approaches, a proportion of protein C (PC) deficiencies remains genetically unresolved. In these cases, deep intronic variation or structural variation could be causal.

Objectives

To identify the causal variation in unrelated patients presenting a PC deficiency in whom no genetic variation was found using conventional exploration.

Patients/Methods

Whole PROC gene was analyzed using next generation sequencing (NGS) in probands with unexplained confirmed decreased PC clot activity after genetic exploration in our centres since 2000. The pathogenic impact of identified candidate variants was assessed using both in silico analysis (MaxEntScan and SpliceAI) and in vitro splicing assay in HeLa and Huh7 cell lines.

Results

Among 2,263 probands, 53 remained unexplained after conventional genetic exploration. Whole PROC gene sequencing was performed in 38/53 probands for which a DNA sample was available. In total, 9 candidate variants from 11 probands (29%) were identified. They corresponded to 7 single nucleotide variants, one 541 bp deletion, and one 1.298 kb balanced inversion. Both the 541 bp deletion and the large balanced inversion disrupted PROC and were considered as pathogenic. Among the seven deep intronic substitutions, splicing functional assay found a deleterious impact (intron retention in mature mRNA or pseudo-exon activation) for four of them: c.237 + 75G > A, c.535 + 936C > T, c.536–95G > A, and c.796 + 49G > T. Thus, these variants were classified as likely pathogenic. Finally, NGS allowed the identification of causal variants in 8/38 patients previously unsolved protein C deficiency (21%).

Conclusion

This study highlights the value of NGS for whole PROC gene sequencing in unexplained protein C deficiency.

Keywords

protein C deficiency - whole gene sequencing - deep intronic variations - structural variants - minigene assay

Authors' Contribution

L.M., P.DM., and Y.J. designed the experiments; L.M., C.R., S.G., I.P., S.L.C., A.V., K.A., and S.B. performed the experiments; L.M., C.R., P.D.M., D.H., S.G., and Y.J. analyzed the data; L.M. and Y.J. drafted the manuscript; P.D.M., D.H., Y.D., C.V., and S.G. helped in revising and editing the manuscript. All the authors approved the manuscript submission.

Publikationsverlauf

Eingereicht: 06. Februar 2025

Angenommen: 28. Juli 2025

Artikel online veröffentlicht:
11. August 2025

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