Introduction

Factor X (FX) is a vitamin K-dependent serine protease precursor synthesized in the liver and plays a central role in blood coagulation [1]. FX is a disulfide-bonded two-chain glycoprotein in the blood plasma with a 17-kDa light chain and a 45-kDa heavy chain [2]. The light chain consists of a -carboxyglutamic acid-rich domain (Gla domain), which is involved in Ca2+-dependent conformational change, and two epidermal growth factor-like domains (EGF-like domain), while the heavy chain contains an activation peptide and a serine protease domain. FX is activated to its active form, activated FX (FXa), by an extrinsic pathway involving complex formation with activated factor VII (FVIIa), tissue factor (TF) and Ca2+, or by an intrinsic pathway involving complex formation with activated factor IX (FIXa), activated factor VIII (FVIIIa), Ca2+ and a phospholipid. Prothrombin is activated by the interaction between FXa and activated factor V (FVa), leading to the progression of a coagulation cascade.

Congenital FX deficiency is a rare bleeding disorder with an incidence of one in one million [3], which follows an autosomal recessive inheritance pattern [4]. According to the Human Gene Mutation Database (HGMD) professional 2019.2 (https://portal.biobase-international.com), 167 different types of genetic mutations had been reported around the world as of August 11, 2019. As for the types of genetic mutation, missense and nonsense mutations caused by single-base substitutions were the most prevalent, accounting for 78% (130 cases) of all mutations. The FX gene (F10), which encodes FX, is a 26.7 kb-long gene located on the long arm of chromosome 13 (13q34), comprising eight exons and seven introns [5]. Congenital FX deficiency is caused by genetic mutations in F10 and develops due to quantitative and qualitative abnormalities of the resulting FX. This pathology can be classified into a deficiency form characterized by decreases in levels of both antigen and activity, and a molecular abnormality form with normal antigen levels, but decreased activity [6]. FX deficiency is one of the most severe coagulation factor deficiencies [7, 8], and presents primarily with umbilical cord bleeding, mucosal bleeding, gastrointestinal bleeding, central nervous system (CNS) bleeding, excessive bleeding after surgery, and recurrent hematoma. While heterozygotes are often asymptomatic, homozygotes and compound heterozygotes are prone to bleeding, with the degree of bleeding correlating with the degree of reduced FX activity.

Agents such as PCCs, plasma-derived FX/FIX concentrate (pd-FX/FIX) and fresh-frozen plasma (FFP) are used to prevent bleeding in congenital FX-deficient patients [9,10,11]. Recently, pd-FX is available as replacement therapy in Europe and USA but not in Japan.

We report herein our experience in preventatively controlling bleeding with regular replacement of 500 units of a PCC, PPSB-HT (Nihon Pharmaceutical, Tokyo, Japan), in a compound heterozygous patient with congenital FX deficiency.

Materials and methods

The proband was a 2-year-old girl (body weight, 14.4 kg) who presented to the pediatric department with a chief complaint of easy bruising. Subcutaneous hematomas (3 ~ 5 cm) were sometimes found on the lower extremity, waist and back. The patient had a history of umbilical cord bleeding at birth. Blood coagulation testing showed a prolonged prothrombin time (PT) of > 40 s and an activated partial thromboplastin time (APTT) of 65.0 s. Marked declines were also seen in FX activity (< 1%) and FX antigen levels (5%). Plasma activities of Factor II, VII, and IX are 84%, 82%, and 81%, respectively. The proband was diagnosed with congenital FX deficiency as a cross-mixing test showed a deficiency pattern. The parents were not consanguineous and the grandmother had a history of bleeding tendency.

Measurement of FX activity and antigen levels

FX activity was measured in a one-stage APTT-based coagulation assay using a THROMBOCHECK Factor X (Sysmex, Hyogo, Japan), and FX antigen levels were assessed using a FX Antigen Elisa kit (Affinity Biologicals, Ontario, Canada) at Nara Medical University.

Sequencing

After obtaining informed consent from the proband and her family, genomic DNA was extracted from peripheral blood leukocytes using a GENERATION Capture Column kit (QIAGEN, Tokyo, Japan). All exons and the exon–intron boundaries of F10 were amplified. KOD-Plus- (TOYOBO, Osaka, Japan) was used for polymerase chain reaction (PCR), and the base sequence of the primer and PCR conditions were adopted from a study conducted by Jayandharan et al. [4]. PCR products were directly sequenced using the dye terminator method with a BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific, Tokyo, Japan). An ABI PRISM 3100 Genetic Analyzer (Thermo Fisher Scientific) was used for genetic analyses.

This study was approved by the Kanazawa University Human Genome and Gene Analysis Research Ethics Committee (Approval no. 135–7).

Regular replacement therapy with PPSB-HT and measurement of FX activity

Regular replacement with 500 units of PPSB-HT at a frequency of once every 2 weeks was planned. FX activity was measured from the proband’s plasma after replacement with 500 units of PPSB-HT. Activity was measured 4 h after replacement, as FX activity peaks approximately 2–4 h after administration. Transfusion of PPSB-HT to the proband was approved through the ethics application process at Musashino Red Cross Hospital (Approval no. 30–9).

Results

Measurement of FX activity and antigen levels

Figure 1 shows FX activity and antigen levels of proband and the family members.

Fig. 1
figure 1

The pedigree of the proband with FX deficiency. The arrow indicated proband. FX activity levels and antigen levels were shown upper and lower, respectively. The proband was suspected of being a compound heterozygote with p.Gly118Asp and p.Gly435Ser. As a result of genetic analysis of family members, the father and elder sister were heterozygous for p.Gly118Asp and the mother was heterozygous for p.Gly435Ser

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Sequencing

Figure 2 shows the results of genetic analysis of the proband and family members. Genetic analysis identified two types of missense mutation, in exon 4 (c.353G >A; p.Gly118Asp) and in exon 8 (c.1303G>A; p.Gly435Ser). The analysis of family members identified heterozygosity, with p.Gly118Asp in the father and elder sister, and p.Gly435Ser in the mother.

Fig. 2
figure 2

Sequencing analysis of the proband and family members. This Fig. shows the result of the genetic analysis of the proband and family members. The sequencing analysis revealed her to have two types of missense mutation, in exon 4 (c.353G>A; p.Gly118Asp) and in exon 8 (c.1303G>A; p.Gly435Ser). The analysis of family members identified heterozygous missense mutations, with p.Gly118Asp in the father and elder sister, and p.Gly435Ser in the mother

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Regular replacement therapy with PPSB-HT and measurement of FX activity

To normalize FX activity and control the bleeding tendencies of the proband, regular replacement therapy was initiated with 500 units of PPSB-HT. As shown in Table 1, PT and APTT were normalized, and FX activity improved to 60% by 4 h after replacement with 500 units of PPSB-HT. The initial plan was one administration every 2 weeks, but additional emergency replacement had to be administered as intra-articular bleeding was detected in the right foot (3–4 cm of swelling) on day 9 after starting replacement therapy. This was the first clear evidence of intra-articular bleeding, and replacements were administered every week thereafter. After shifting to replacement therapy once a week, no bleeding tendencies or new bruises have been observed. Moreover, PT-INR and APTT, which were 9.1 and 65.0 s at diagnosis, have been improved to 4.7 and 50.6 s after 8 months of the regular replacement therapy. FX inhibitor assay results 8 months after starting replacement therapy were negative.

Table 1 Blood coagulation tests of the proband at diagnosis and after administration with PPSB-HT
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Discussion

Genetic analysis of the proband identified two types of missense mutations (c.353G>A, p.Gly118Asp and c.1303G>A, p.Gly435Ser), demonstrating compound heterozygosity of the proband for congenital FX deficiency. The p.Gly118Asp mutation was reported by Flora et al. in 2001, and Matsuo et al. reported the p.Gly435Ser mutation in 2016.

Based on previous reports, the following is the suspected mechanism of amino acid substitution at the site of each mutation that triggers FX deficiency. Gly118 is located within the EGF-1 domain. The EGF-1 domain is in a region involved in Ca2+ binding and in the structural maintenance of FXa. Gly118 is a highly conserved amino acid among vitamin K-dependent coagulation factors (VKDCF) and among mammals (Fig. 3), and plays a crucial role in protein folding [5]. Gly118Asp mutation may have caused the decline in FX activity by causing protein misfolding leading to impaired secretion. However, more detailed experiments such as functional analysis of a mutant FX protein should be conducted to confirm this hypothesis. On the other hand, p.Gly435Ser is a missense mutation within the serine protease domain. Gly435 is highly conserved among VKDCF and among mammals (Fig. 3) and completely conserved in the serine protease family, and thus appears to represent a particularly important amino acid in the catalytic region [12]. Gly435Ser mutation may have caused the decline in FX activity by destabilizing the three-dimensional structure of the protein in the catalytic region, leading to impaired secretion.

Fig. 3
figure 3

Comparison of amino acid alignments around Gly118 and Gly435. Gly118 and Gly435 are highly conserved among vitamin K-dependent coagulation factors and among mammals

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As rare coagulation disorders (RCDs) such as congenital FX deficiency are not encountered very often, specialist physicians select the course of treatment based on experience, due to the lack of epidemiological findings and clinical results [13]. The half-life of FX in the blood is approximately 40–60 h, relatively longer than other coagulation factors, and a FX level of 10–20% is considered to be needed for sufficient hemostasis [14, 15]. Agents such as PCCs, pd-FX/FIX, and FFP are used in the treatment of congenital FX-deficient patients. However, FFP is known to have side-effects such as circulatory overload, anaphylactic shock and transfusion-related acute lung injury (TRALI) [16, 17]. When administering pd-FX/FIX concentrate and PCCs, there is a risk of thrombosis from the accumulation of non-specific coagulation factors other than FX [15, 18]. Currently available PCCs contain various anti-coagulants (such as heparin, antithrombin, protein C, protein S, and protein Z) to minimize this risk. However, for FX deficiency, coagulation factor concentrate containing only FX should ideally be used [19]. In 2016, a high-purity pd-FX (Bio Products Laboratory, Elstree, UK) was developed as a therapeutic agent for congenital FX deficiency [3] and has since been approved for use in the United States and Europe. While FX replacement with pd-FX is recommended for congenital FX deficiency patients, the use of pd-FX has not yet been approved in Japan. FX replacement was thus performed using PCCs as a substitute for pd-FX. Cases have been reported of PCCs and pd-FX being used in FX replacement therapy during bleeding, but no reports have described PCCs usage in regular replacement therapy.

In the present study, PPSB-HT was used as a substitute for pd-FX in regular replacement therapy, and proved effective in preventative control of bleeding tendency in congenital FX deficiency. As shown in Table 1, PT and APTT were normalized, and FX activity improved by 4 h after administration. Plasma activities of factor II, VII, and IX increased slightly by contrast with before administration. Although PT and APTT were prolonged and FX activity reduced below 1% at 1 week after the first administration, her bleeding tendency disappeared clinically. Even more surprising, PT-INR and APTT gradually were shortened as the regular replacement therapy continued. In general, the amount of FX replacement increases as the child grows. It seems that the improvement in the bleeding tendency by the regular replacement therapy has reduced the consumption of FX. Therefore, it is considered that the administration every week was effective in improving the bleeding tendency in this patient.

In the future, we need to examine the replacement dosage and frequency to account for increased body weight during the growth period, and we hope to elucidate the optimal replacement dosage and frequency through the accumulation of more cases. As FX replacement consequently results in excessive administration of factor II, VII, and IX, possibly increasing the risk of thrombosis due to hypercoagulability, monitoring by means such as measurement of coagulation factor activities is important. It is also helpful to try to exclude thrombosis by measurement of coagulation activity markers such as thrombin-antithrombin complex, prothrombin fragment 1 + 2 and soluble fibrin in plasma. Recently, comprehensive coagulation assays, such as clot waveform analysis (CWA), Thrombelastography and thrombin generation assay (TGA), have been developed to assess physiological hemostasis. Therefore, these assays could provide valuable information on coagulation potential in the patient with the regular replacement therapy of a PCC. Monitoring for the emergence of inhibitors is also essential.