Whole PROC Gene Sequencing to Explain Genetically Unresolved Protein C Deficiencies

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.To identify the causal variation in unrelated patients presenting a PC deficiency in whom no genetic variation was found using conventional exploration.Whole <i>PROC</i> 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 <i>in silico</i> analysis (MaxEntScan and SpliceAI) and in vitro splicing assay in HeLa and Huh7 cell lines.Among 2,263 probands, 53 remained unexplained after conventional genetic exploration. Whole <i>PROC</i> 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 <i>PROC</i> 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%).This study highlights the value of NGS for whole <i>PROC</i> gene sequencing in unexplained protein C deficiency.