Analysis of PROS1 mutations and clinical characteristics in three Chinese families with hereditary protein S deficiency.

We report three heterozygous PROS1 mutations that caused type I protein S deficiency in three unrelated Chinese families. We measured protein S activity and antigen levels for all participants, screened them for mutations in the PROS1 gene. And we employed the calibrated automated thrombin generation (CAT) method to investigate thrombin generation. Numerous bioinformatics tools were utilized to analyze the conservation, pathogenicity of mutation, and spatial structure of the protein S. Phenotyping analysis indicated that all three probands exhibited simultaneous reduced levels of PS:A, TPS:Ag, and FPS:Ag. Genetic testing revealed that proband A harbored a heterozygous c.458_458delA (p.Lys153Serfs*6) mutation in exon 5, proband B carried a heterozygous c.1687C>T (p.Gln563stop) mutation in exon 14, and proband C exhibited a heterozygous c.200A>C (p.Glu67Ala) mutation in exon 2. Bioinformatic analysis predicted that the p.Lys153Serfs*6 frameshift mutation and the p.Gln563stop nonsense mutation in the protein S were classified as "disease-causing." The identification of the novel mutation p.Lys153Serfs*6 in PROS1 enriches the Human Genome Database. Our research suggests that these three mutations (p.Lys153Serfs*6, p.Gln563stop, and p.Glu67Ala) are possibly responsible for the decreased level of protein S in the three families. Furthermore, the evidence also supports the notion that individuals who are asymptomatic but have a family history of PSD can benefit from genetic analysis of the PROS1 gene.

The clinical and genetic landscape of early-onset thrombophilia in Japan.

OBJECTIVES: To determine the optimal management for early-onset thrombophilia (EOT), the genetic and clinical features of protein C (PC)-, protein S (PS)-, or antithrombin (AT)-deficient patients of ≤20 years of age were studied in Japan. METHODS/RESULTS: Clinical and genetic information of all genetically diagnosed cases was collected through the prospective, retrospective study, and literature review. One-hundred-one patients had PC (n = 55), PS (n = 29), or AT deficiency (n = 18). One overlapping case had PC- and PS-monoallelic variant. Fifty-five PC-deficient patients (54%) had 26 monoallelic or 29 biallelic variant(s), and 29 (29%) PS-deficient patients had 20 monoallelic or nine biallelic variant(s). None of the patients had AT-biallelic variants. The frequent low-risk allele p.K193del (PC-Tottori) was found in five patients with monoallelic (19%) but not 29 with biallelic variant(s). The most common low-risk allele p.K196E (PS-Tokushima) was found in five with monoallelic (25%) and six with biallelic variant(s) (67%). One exceptional de novo PC variant was found in 32 families with EOT. Only five parents had a history of thromboembolism. Thrombosis concurrently developed in three mother-newborn pairs (two PC deficiency and one AT deficiency). The prospective cohort revealed the outcomes of 35 patients: three deaths with PC deficiency and 20 complication-free survivors. Neurological complications were more frequently found in patients with PC-biallelic variants than those with PC-, PS-, or AT-monoallelic variants (73% vs. 24%, p = .019). CONCLUSIONS: We demonstrate the need for elective screening for EOT targeting PC deficiency in Japan. Early prenatal diagnosis of PC deficiency in mother-infant pairs may prevent perinatal thrombosis in them.

PROS1 variant c.1574C>T p.Ala525Val causes portal vein thrombosis with protein S deficiency.

BACKGROUND: Protein S (PS) is a vitamin K-dependent plasma glycoprotein, and the deficiency of PS increases the risk of venous thromboembolism (VTE). PS deficiency has been found in 1.5-7% of selected groups of thrombophilic patients. However, the reported PS deficiency patients with portal vein thrombosis are scarce. CASE REPORT AND RESULTS: Our case described a 60-year-old male patient presented portal vein thrombosis with protein S deficiency. Imaging findings of the patient revealed extensive thrombosis involving the portal vein and superior mesenteric vein. His medical history revealed lower extremity venous thrombosis 10 years ago. The level of PS activity was greatly reduced (14%, reference: 55-130%). Acquired thrombophilia caused by antiphospholipid syndrome, hyperhomocysteinemia, or malignancy were excluded. Whole exome sequencing revealed a heterozygous missense variation c.1574C>T, p.Ala525Val in the PROS1 gene. The in-silico analysis of the variant was performed by SIFT and PolyPhen-2. The results showed that the variant is a pathogenic and likely pathogenic variation respectively (SIFT, -3.404; PolyPhen-2, 0.892), the amino acid substitution A525V is presumed to result in unstable PS protein which is degraded intracellularly. Mutation site of the proband and his family members was validated by Sanger sequencing. CONCLUSION: According to the clinical manifestation, imaging findings, protein S level, and the genetic results, a diagnosis of portal vein thrombosis with PS deficiency was made. To the best of our knowledge, our case is the second reported PS deficiency patient caused by PROS1 c.1574C>T, p.Ala525Val variant in Asia, and the case is also the only reported case with PROS1 c.1574C>T, p.Ala525Val variant presents portal vein thrombosis.

A Case of a Pediatric Patient With Protein S Heerlen Polymorphism and Deep Venous Thrombosis.

Hereditary protein S (PS) deficiency is a rare autosomal dominant disorder with increased risk of venous thromboembolism. The PS Heerlen polymorphism at codon 501 of the PROS1 gene is considered a variant of uncertain significance. It has since been shown that PS Heerlen has a reduced half-life, resulting in reduced levels of free PS. We report a case of an adolescent female with May Thurner syndrome and heterozygous PS Heerlen mutation resulting in a mild PS deficiency and venous thromboembolism. With this nonmodifiable risk factor, the patient received prolonged anticoagulation with strong consideration for lifelong prophylaxis.

Otogenic lateral sinus thrombosis in children: proposal of an experience-based treatment flowchart.

PURPOSE: To describe the prevalent clinical, laboratory, and radiological features of otogenic lateral sinus thrombosis (OLST) in children; to identify clinical predictors of outcome; to propose a management algorithm derived from experience. METHODS: A retrospective review was conducted of the clinical records of patients with OLST, treated in a single tertiary care referral center for pediatric disease from 2006 to 2017. The inclusion criteria were pediatric age (0-16 years) and OLST diagnosis confirmed by a pre- and post-contrast CT or venography-MRI scan. Primary outcome measures were early (1-2 months) and late (6 months) sinus recanalization assessed by means of neuroimaging. RESULTS: Twenty-five patients (8 females and 17 males; mean age = 6 ± 3 years) were included. A genetic abnormality associated with thrombophilia was found in 24 (96%) patients. At diagnosis, anticoagulant treatment with low-molecular-weight heparin (LMWH) was started in all subjects, while surgical treatment (mastoidectomy and tympanostomy tube insertion) was performed in 16/25 (64%) patients. Follow-up neuroimaging showed lateral sinus recanalization in 12/25 (48%) patients after 1-2 months and in 17/25 (68%) after 6 months. At multivariate logistic regression analysis, no significant predictors of the early and late neuroradiological outcome were found. CONCLUSIONS: All children with OLST should be screened for thrombophilia to decide on treatment duration and to assess the need for future antithrombotic prophylaxis. Immediately after diagnosis, anticoagulant treatment with LMWH should be started according to the international guidelines. Instead, our experience suggests that surgical treatment should not be indicated in all patients, but decided on a case-to-case basis.

Identification of reference miRNAs in plasma useful for the study of oestrogen-responsive miRNAs associated with acquired Protein S deficiency in pregnancy.

BACKGROUND: Accumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users. In order to identify other oestrogen-responsive miRNAs, including miR-494, that may be indicative of increased thrombotic risk in plasma, we used nanoString analysis to identify robust and stable endogenous reference miRNAs for the study of oestrogen-responsive miRNAs in plasma. RESULTS: We compared the plasma miRNA expression profile of individuals with: (1) Low circulating oestrogens (healthy men and non-pregnant women not taking oral contraceptives), (2) High circulating synthetic oestrogens, (women taking oral contraceptives) and (3) High circulating natural oestrogens (pregnant females >14 weeks gestation). From the nanoString analyses, 11 candidate reference miRNAs which exhibited high counts and not significantly differentially expressed between groups were selected for validation using realtime quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (DDPCR) in pooled plasma samples, and the stability of their expression evaluated using NormFinder and BestKeeper algorithms. Four miRNAs (miR-25-5p, miR-188-5p, miR-222-3p and miR-520f) demonstrated detectable stable expression between groups and were further analysed by RT-qPCR in individual plasma samples, where miR-188-5p and miR-222-3p expression were identified as a stable pair of reference genes. The miRNA reference panel consisting of synthetic spike-ins cel-miR-39 and ath-miR159a, and reference miRNAs, miR-188-5p and miR-222-3p was useful in evaluating fold-change of the pregnancy-associated miRNA, miR-141-3p, between groups. CONCLUSION: The miRNA reference panel will be useful for normalising qPCR data comparing miRNA expression between men and women, non-pregnant and pregnant females, and the potential effects of endogenous and synthetic oestrogens on plasma miRNA expression.

Venous thromboembolism associated with protein S deficiency due to Arg451* mutation in PROS1 gene: a case report and a literature review.

Protein S (PS) is a vitaminK-dependent glycoproteinwhich plays an important role in the regulation of blood coagulation. PS deficiency has been found in 1.5-7% of thrombophilic patients. Here, we report the first Polish case with PS deficiency caused by the p.Arg451* in the PROS1 gene detected in a 21-year-old man with trauma-induced venous thromboembolism. To our knowledge, we provided the review of all the available data on this mutation (a total of 56 cases). The proband, his mother and his sister were screened for thrombophilia. To elucidate genetic background of PS deficiency, all PROS1 genes were subjected to direct sequencing. The free PS levels were 35% in the proband, 21% in the proband's mother and 28% in the proband's sister and their PS total levels were 37.1, 47.5 and 55.1%, respectively. Type I PS deficiency was diagnosed. In all patients, genetic analysis revealed the presence of heterozygous nonsense mutation (c.1351C>T; p.Arg451*) located in exon 12 of PROS1 gene. This mutation interrupts the reading frame by premature termination codon at position 451 and may lead to the production of truncated protein. The present case combined with the review of the literature suggests that p.Arg451* in the PROS1 gene mainly leads to clinically evident thrombosis following trauma, surgery or serious comorbidities especially malignancy.

Genotypes and phenotypes of protein S deficiency in Thai children with thromboembolism.

The prevalence of protein S (PS) deficiency in Asian patients with venous thromboembolism is around 8-30%, higher than that in Caucasian populations. The present study reports the genotypes (including one novel mutation) and phenotypes of children with PS deficiency at a tertiary care institute. A total of six patients were included, three with arterial ischemic stroke, two with cerebral venous sinus thrombosis, and one with deep vein thrombosis. PS mutations were identified in four patients: p.R355C, p.G336D, p.E67A, and p.N188KfsX9. p.N188KfsX9 is a novel mutation with less than 20% PS activity noted in heterozygotes.

Ertapenem-Induced Acute Interstitial Nephritis (AIN) in a Case of Protein S Deficiency and Factor V Leiden Mutation with Deep Vein Thrombosis.

We present a case of 58 years old male patient, who presented with high fever for which injection Ertapenem was started empirically at Dubai hospital. Patient was a known case of Deep vein thrombosis of left leg since 5 years on warfarin therapy. Patient came to India for high fever and further management. Patient developed proteinuria with high creatinine and urinary abnormalities. Renal biopsy revealed acute interstitial nephritis (AIN). In addition, he was diagnosed to have protein S deficiency with Factor V Leiden mutation.

Hypercoagulable States: What the Oral Surgeon Needs to Know.

Thrombophilia or hypercoagulable conditions can be thought of as either inherited or acquired. The inherited disorders include deficiencies of antithrombin, protein C, or protein S or the common disorders of factor V Leiden and prothrombin G20210A gene mutation. All these disorders are inherited as autosomal dominant and predispose individuals primarily to venous thrombosis. Acquired thrombophilic conditions are seen in individuals with cancer, phospholipid antibodies, and a whole host of other conditions that alter endothelial function, change blood levels of coagulant or anticoagulant proteins, activate platelets, or have other effects on coagulation proteins, platelet function, or the endothelium.

Antithrombotic prophylaxis in a patient with nephrotic syndrome and congenital protein S deficiency.

BACKGROUND: Nephrotic syndrome confers an acquired prothrombotic phenotype due to the urinary loss of anticoagulant proteins.Patients with reactivation of nephrotic syndrome may develop thrombosis. CASE PRESENTATION: We report the case of a life-threatening cerebral venous thrombosis in a 13 year-old boy affected by a relapse of nephrotic syndrome during a P. aeruginosa otitis/mastoiditis. Due to the worsening general conditions and the severe neurological impairment, a course of systemic thrombolysis was successfully administered, followed by anticoagulant therapy. In the present case severe inherited thrombophilia (inherited dysfunctional protein S deficiency) was identified as an important additional risk factors for thrombosis. CONCLUSIONS: A careful evalutaion of risk factos for thrombosi during reactivation of nephrotic syndrome include measurement of plasma anticaogulant proteins. When low, antithrombotic prophylaxis with heparin should be considered to prevent thrombotic episodes.

Prevalence of thrombophilia according to age at the first manifestation of venous thromboembolism: results from the MAISTHRO registry.

Thrombophilia is a well-established risk factor for a venous thromboembolic event (VTE), and it has been proposed that hereditary thrombophilia may substantially contribute to the development of VTE in young patients. We aimed to analyse the prevalence of thrombophilia with special regard to the age of VTE manifestation. The study cohort consisted of 1490 patients (58% females) with a median age 43 years at the time of their first VTE. At least one thrombophilic disorder was identified in 50·1% of patients. The probability of detecting a hereditary thrombophilia declined significantly with advancing age (from 49·3% in patients aged 20 years and younger to 21·9% in patients over the age of 70 years; P < 0·001). This may be primarily attributed to the decreasing frequencies of the F5 R506Q (factor V Leiden) mutation and deficiencies of protein C or protein S with older age at the time of the initial VTE event. Moreover, thrombophilia was more prevalent in unprovoked compared with risk-associated VTE (57·7% vs. 47·7%; P = 0·001). The decline in the prevalence of hereditary thrombophilia with older ages supports the use of a selected thrombophilia screening strategy dependent on age and the presence or absence of additional VTE risk factors.

Micro-ribonucleic Acid 494 regulation of protein S expression.

BACKGROUND: Acquired protein S (PS) deficiency is highly associated with elevated circulating estrogen levels resulting from pregnancy, oral contraceptives, and estrogen replacement therapy; however, the mechanism of estrogen-mediated acquired PS deficiency remains poorly understood. Increasing evidence indicates that estrogen receptor signaling can indirectly modulate the expression of target genes at the post-transcriptional level by modulating the expression of microRNAs (miRNAs), and miRNAs have also been demonstrated to be involved in the regulation of hemostasis. OBJECTIVES: To investigate the mechanism of estrogen-mediated downregulation of PROS1 expression by the microRNA miR-494. METHODS: Computational analyses of the PROS1 3'-untranslated region (UTR) were performed to identify putative miRNA-binding sites, and direct targeting of the PROS1 3'-UTR by miR-494 was determined with dual luciferase reporter assays in HuH-7 cells. Reporter vectors containing the PROS1 3'-UTR sequence with deleted miR-494-binding sites were also analyzed with luciferase reporter assays. The effects of estrogen on miR-494 and PROS1 mRNA levels in HuH-7 cells were determined by quantitative real-time PCR, and estrogen-mediated changes to secreted PS levels in culture supernatant of HuH-7 cells were measured with an ELISA. RESULTS: The PROS1 3'-UTR sequence contains three putative miR-494-binding sites. miR-494 directly targets PROS1, and miR-494 levels are upregulated following estrogen treatment in HuH-7 liver cells in association with downregulated PROS1 mRNA and PS levels. CONCLUSIONS: The results from this study provide the first evidence for miRNA downregulation of PROS1 by miR-494, and suggest that miR-494 is involved in the mechanism of estrogen-mediated downregulation of PS expression.

[Case of cerebral venous thrombosis secondary to Protein S gene abnormality first diagnosed as aseptic meningitis].

A 28-year-old man admitted to our hospital because of severe neck pain, headache, fever and vomiting. He was alert and had no neurological deficit except for nuchal stiffness. Cerebrospinal fluid (CSF) examination showed elevated mononuclear cell counts (68/mm(3)) and protein levels (300 mg/dl). He was diagnosed as having aseptic meningitis and was thereby treated. Two days later, he manifested seizure and on the next day, severe cerebral hemorrhage occurred in the left parietal lobe and decompression surgery was performed. Cerebral venous thrombosis was strongly indicated by operative findings, cerebral angiography and retrospective assessment of MRI images. Genetic testing revealed a novel mutation (p.Cys449Tyr) combined by Protein S Tokushima mutation (p.Lys196Glu) which is frequently observed in Japanese. Possibility of CVT should be noted, even when a patient exhibits clinical symptoms and CSF findings compatible with a diagnosis of aseptic meningitis.

Hypercoagulable states.

Hypercoagulable states can be inherited or acquired. Inherited hypercoagulable states can be caused by a loss of function of natural anticoagulant pathways or a gain of function in procoagulant pathways. Acquired hypercoagulable risk factors include a prior history of thrombosis, obesity, pregnancy, cancer and its treatment, antiphospholipid antibody syndrome, heparin-induced thrombocytopenia, and myeloproliferative disorders. Inherited hypercoagulable states combine with acquired risk factors to establish the intrinsic risk of venous thromboembolism for each individual. Venous thromboembolism occurs when the risk exceeds a critical threshold. Often a triggering factor, such as surgery, pregnancy, or estrogen therapy, is required to increase the risk above this critical threshold.

Venous thromboembolism in Asia--an unrecognised and under-treated problem?

Venous thromboembolism (VTE) has been perceived for a long time to be less common in Asian populations, particularly in the Far East, than in Western populations. Generally, thromboprophylaxis is not implemented as frequently as it should be in high-risk patients. However, recent prospective studies undertaken in Asian countries have demonstrated higher rates of VTE after major surgery and in medical wards, approaching those observed in Western populations. Risk factors for VTE are not different in Asian patients from those of Western patients with the exception of thrombophilic mutations. Deficiencies of the natural anticoagulants (protein S, protein C, and antithrombin) are the predominant thrombophilias in Asia whereas factor V Leiden and prothrombin G20210A gene mutation are not found or rarely reported. Further large well-designed clinical studies are needed to evaluate the magnitude of the risk of VTE and the appropriate use of thromboprophylaxis in different clinical situations.

[Recurrent deep vein thrombosis caused by heterozygous missense mutation of the protein S gene: genetic analysis of a case].

OBJECTIVE: To analyze the molecular pathogenesis of protein S deficiency in an adolescent case of recurrent deep vein thrombosis (DVT). METHODS: Blood samples from the patient and his family members were collected for detection of the coagulation parameters by one-step clotting method, and the protein S (PS) and protein C activities were measured by a chromogenic assay. Enzyme-linked immunosorbent assay was employed for detecting the levels of free PS antigen. All the exons and exon-intron boundaries of the patients PS gene were amplified using PCR and analyzed by direct sequencing. RESULTS: As carriers of hereditary PS deficiency, both the patient and his father showed a heterozygous C82792T point mutation in the 10th exon of their PS gene which resulted in the substitution of arginine314 by cysteine in the polypeptide chain of PS protein. CONCLUSION: Recurrence of DVT in this patient is the result of hereditary PS deficiency caused by a novel heterozygous missense mutation in the PS gene.

Protein S deficiency with a PROS1 gene mutation in a patient presenting with mesenteric venous thrombosis following total colectomy.

Protein S deficiency (PSD) is an inherited or acquired disorder associated with an increased risk of thrombosis. We describe the unusual case of a previously healthy 45-year-old man with PSD. The patient was admitted for work-up of hematochezia and was diagnosed with colorectal cancer. Preoperative evaluation was unremarkable, and he subsequently underwent an uneventful laparoscopic total colectomy. The next day, he complained of chest tightness, and a computed tomography enterography revealed acute thrombosis throughout the entire superior mesenteric vein. Further laboratory evaluation revealed decreased free protein S levels. Polymerase chain reaction and direct sequencing of the PROS1 gene revealed a heterozygous transition (NM_000313.3 c.1907A > G), resulting in an amino acid change (p.Tyr636Cys). Although several case reports have described mesenteric venous thrombosis (MVT) in individuals with PSD, most have just reported decreases in the activity or concentration of protein S. We confirmed a nucleotide alteration of PROS1 associated with PSD. This patient, who had hereditary thrombophilia, may have developed MVT after transient exposure to abdominal surgery. We may need to implement more comprehensive coagulation testing prior to surgery considering the prevalence and possible risk of thromboembolic event.

Hereditary thrombophilia in an unselected cohort of venous thrombosis patients in Singapore.

AIM: Hereditary thrombophilic markers are commonly screened among patients diagnosed as having venous thromboembolism, but optimal patient selection and the goals of screening may differ between populations. Determining the patterns of hereditary thrombophilia may improve screening strategies. METHOD: An unselected cohort of venous thromboembolism patients in three tertiary institutions in Singapore was prospectively tested for the prevalence of deficiencies of protein C, protein S, antithrombin III, factor V Leiden and prothrombin 20210 gene mutations. RESULTS: Among 384 patients screened, the prevalences of protein S, protein C and antithrombin III were 9.20%, 1.18% and 4.19% respectively. Only one patient was positive for the factor V Leiden mutation and none tested positive for the prothrombin 20210 gene mutation. At least 1 in 9 patients (11.52%, 95% CI 8.20 to 15.93) will test positive for one of the above markers in an unselected group of 269 patients who completed all tests. The exclusion of patients with clinical risk factors did not improve the detection rates, in comparison with those with obvious provoking clinical risk factors (11.72%, 95% CI 7.36 to 18.06 vs 11.29%, 95% CI 6.73 to 18.18). When upper age limits were set for thrombophilia screening by decades, a statistical difference in the likelihood of a positive thrombophilia screen between younger and older patient was seen for patients below 40 (p<0.001). CONCLUSION: In Singapore and countries with similar demographics, hereditary thrombophilia screening should be confined to testing for protein C, protein S and antithrombin III.