Pathogenic variants of PROC gene caused type I activity deficiency in a familial Chinese venous thrombosis

Abstract Pathogenic mutation of protein C (PROC) gene results into the deficiency of PROC activity. This study aimed to identify the pathogenic genetic variants and to explore the functional consequence in Chinese familial venous thrombosis (VTE). Whole exome sequencing was performed to identify the pathogenic variants of anticoagulant factors. Serum coagulation and anti‐coagulation factors activity were assayed to evaluate the genetic association. Functional study of PROC antigen secretion deficiency was conducted in VTE subjects and in vitro cell lines. One rare pathogenic variant (p.Ala178Pro) was identified in the four VTE subjects but not in the normal subjects from the family. An inframeshift variant (rs199469469) was also identified in a paediatric subject of the pedigree. Further evaluation of serum PROC activity levels in p.Ala178Pro variants VTE carriers showed significantly lower PROC activity compared to non‐carriers. Furthermore, in vitro study showed that the p.Ala178Pro mutant cells had a consistent reduction in concentration of PROC antigen. In conclusions, our study demonstrated the pathogenic variant (p.Ala178Pro) contributed to PROC type I activity deficiency, which may be due to decreased secretion of PROC.

other coagulation factors, which are major contributors for the susceptibility to VTE. 3 PROC is a vitamin K-dependent serine protease zymogen in plasma, and activated protein C (APC) exerts its anticoagulant function through inactivation of the blood coagulation factors FVa and FVIIIa. 4 Based on the functional and immunological PROC assays, PROC deficiency can be divided into two types. Type I deficiency is characterized by a reduction in the antigen concentration and activity function, whereas type II deficiency is featured by normal or increased antigen concentration but reduced activity. 5 Homozygous or compound heterozygous PROC deficiency usually leads to VTE. Previous studies already identified hundreds of PROC and PROS1 mutations; however, the mutation pattern is different among Western and Asian populations. 6 These variants in the individuals only partly contributed to the PROC activity deficiencies, suggesting other pathogenic mutations may be involved. 7 The aim of the study is to investigate whether familial VTE patients with low APC level would carry PROC or PROS1 genetic variants and to evaluate the association among VTE and PROC or PROS1 deficiencies in the Chinese population. Identification of hereditary thrombophilic genetic factors would enable prognosis, prevention and genetic counselling for VTE patients and family members.

| Subjects
A total of 212 sporadic VTE patients and one family with four VTE patients and three normal siblings were recruited during December 2013 to June 2018 in the Shenzhen People's Hospital. 8 The PTE patients were diagnosed according to the criteria released by European Society of Cardiology (ESC) in 2014. 9 In addition, a total of 196 sporadic control subjects were recruited. The recruited criterions of healthy controls were same as we described before. 10 Informed consent was obtained from all the patients and healthy individuals before enrolled in the study. All the research procedures are approved by the Ethics Committee of Shenzhen People's Hospital. All procedures performed in studies involving human participants were in accordance with the 1964 Declaration of Helsinki ethical standards.

| Whole exome sequencing and variants validation
Qualified genomic DNA was extracted from the whole blood, and capture-based target enrichment was performed by Agilent SureSelect All Exon V5 capture kit according to the manufacturer's protocols. Massively parallel sequencing was performed by HiSeq4000 platform (Illumina). All variants including nonsynonymous, insertion or deletion and non-coding variants occurring in the genomic region among PROC were evaluated. The variants with MAF and function were annotated by databases of gnomeAD, avsnp150, and so on. Tolerant (SIFT), Polyphen2 HumVar and CADD (combined annotation dependent depletion) were used to assess the pathogenesis of these variants. Sanger sequencing was performed to validate all candidate variants in the VTE familial cohort.

| Serum coagulation and anti-coagulation factors activity assays
Blood samples of VTE were collected into Na-citrate containing va-

| Cell culture
The effect of p.Ala178Pro PROC antigen secretion and synthe-

| In vitro functional assays
The cell lysates and culture conditioned media were harvested at 72 hours after transfection. Briefly, nine well culture media were mixed and then freeze concentrated for improving the PROC antigen since the concentration is too low to be detectable. The PROC antigen and expression levels in cell lysates were normalized against the transfection efficiency. The secreted PROC antigen concentrations in culture media were measured by ELISA kit (Abnova) according to the manufacturer's protocol. SPSS 13.0 (IBM) was used for statistical analysis. Independentsamples t test was conducted among the case-control groups.

| Statistical analysis
The results with P value less than .05 were considered statistically significant.

| Patient characteristics
In the pedigree of the 3-generation Chinese family, the proband showed phenotype of PTE with very low PROC activity. Among the family members, three subjects had PTE (IIa, IIb and IId), one had cerebral infarction (Ia) and one had deep vein thrombosis and PTE (IIIb). The patient of IIa died of PTE. Three normal familial members (Ib, IIe, IIf) did not showed any clinical thrombotic phenotype ( Figure 1A).

| Pathogenic variants were identified by whole exome sequencing
Whole exome sequencing was conducted in the family subjects to identify the inherited genetic factors. All coding variants presented in the coding region among PROC and PROS1 were identified. The variants with MAF geater than 5% in databases (gnomeAD) were considered as common variants. After filtering within-house bioinformatics pipeline, 11 variants were identified in PROC and only two variants (NM_000312: p.A178P, p.K193del) are located in exon region with rare frequency (Table S1). The variant allele minor the PHRED value is 13, which indicates that these are predicted to be the 10% most deleterious substitution. In the family members, only two subjects (IIb and IId) showed protein S deficiency, while we did not find any coding variant in PROS1 and PROCR (PROC receptor) among the VTE cases ( Table 1). The functional deficiency consequences of PROS1 might be caused by the regulatory effects of other unknown genetic changes. FDP and PLGA were found to be within normal ranges in all the patients.

| PROC activity deficiency analysis in variants carrying subjects
The activities of serum coagulation and anti-coagulation factors were measured to check the association between genetic defects and clin-  (Table 1).

| Effect of p.Ala178Pro mutation on PROC antigen secretion and synthesis
To characterize the influence of the p.Ala178Pro mutation on PROC antigen secretion and synthesis, the PROC secretion in VTE subjects and in vitro cells was detected. The intracellular protein expression of PROC is similar in each VTE individual. The ELISA results showed secretion of serum PROC was reduced by 31.8%-55% in the VTE subjects compared with control ( Figure 1C). The mean concentration of PROC antigen decreased to 58% in VTE subjects. Interestingly, the serum PROC antigen secretion reduction is consistent with the reduction of APC, such as IIb and IId showed the lowest serum PROC secretion and APC. In vitro study demonstrated that the mRNA expression and intracellular protein expression of PROC were not significantly different between wild-type and mutant cell lysates.
However, PROC secretion in conditioned media from mutant cells reduced by 25% in ECV-304 and by 68% in HEK293T cells, respectively, compared with wild-type cells ( Figure 1D).

| D ISCUSS I ON
In this study, we identified a rare pathogenic PROC variant  the reduced anticoagulant activity of PROC was associated with other two nearby mutations of p.Arg189Trp and p.Lys193del in the Chinese population. 12 The genetic mutations of p.Ala178Pro that would impair PROC folding or degradation in endoplasmic reticulum or Golgi apparatus which may lead to the APC generation decreased.
To further elucidate the association between genetic mutations and PROC deficiency, the protein intracellular expression and extracellular secretion were examined in wild-type and mutant cells.
Previous in vitro study showed that most of missense mutations of PROC resulted in a decreased secretion of PROC, which was resulted from aberrant protein structure or changes in substrate recognition sites. 13

ACK N OWLED G EM ENTS
We would like to express our thanks to the support from Shenzhen

Public Service Platform on Tumor Precision Medicine and Molecular
Diagnosis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S ' CO NTR I B UTI O N S
YY and YF prepared the project proposal and study design, super-