Hereditary Protein Research Articles

Rivaroxaban Causes Missed Diagnosis of Protein S Deficiency but Not of Activated Protein C Resistance (Factor V Leiden).

- Rivaroxaban causes a false increase in activated protein C resistance (APCR) ratios and protein S activity. - To investigate whether this increase masks a diagnosis of factor V Leiden (FVL) or protein S deficiency in a "real-world" population of patients undergoing rivaroxaban treatment and hypercoagulation testing. - During a 2.5-year period, we compared 4 groups of patients (n = 60): FVL heterozygous (FVL-HET)/taking rivaroxaban, wild-type/taking rivaroxaban, FVL-HET/no rivaroxaban, and normal APCR/no rivaroxaban. Patients taking rivaroxaban were tested for protein S functional activity and free antigen (n = 32). - The FVL-HET patients taking rivaroxaban had lower APCR ratios than wild-type patients ( P < .001). For FVL-HET patients taking rivaroxaban, mean APCR was 1.75 ± 0.12, versus 1.64 ± 0.3 in FVL-HET patients not taking rivaroxaban ( P = .005). Activated protein C resistance in FVL-HET patients fell more than 3 SDs below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite rivaroxaban. In contrast, rivaroxaban falsely elevated functional protein S activity, regardless of the presence or absence of FVL ( P < .001). A total of 4 of 32 patients (12.5%) had low free protein S antigen (range, 58%-67%), whereas their functional protein S activity appeared normal (range 75%-130%). Rivaroxaban would have caused a missed diagnosis of all cases of protein S deficiency during the study if testing relied on the protein S activity assay alone. - Despite rivaroxaban treatment, APCR testing can distinguish FVL-HET from normal patients, rendering indiscriminate FVL DNA testing of all patients on rivaroxaban unnecessary. Free protein S should be tested in patients taking rivaroxaban to exclude hereditary protein S deficiency.

Protein S Heerlen mutation heterozygosity is associated with venous thrombosis risk

Hereditary Protein S (PS) deficiency is a rare coagulation disorder associated with an increased risk of venous thrombosis (VT). The PS Heerlen (PSH) mutation is a rare S501P mutation that was initially considered to be a neutral polymorphism. However, it has been later shown that PSH has a reduced half-life in vivo which may explain the association of PSH heterozygosity with mildly reduced levels of plasma free PS (FPS). Whether the risk of VT is increased in PSH carriers remains unknown. We analyzed the association of PSH (rs121918472 A/G) with VT in 4,173 VT patients and 5,970 healthy individuals from four independent case-control studies. Quantitative determination of FPS levels was performed in a subsample of 1257 VT patients. In the investigated populations, the AG genotype was associated with an increased VT risk of 6.57 [4.06–10.64] (p = 1.73 10−14). In VT patients in whom PS deficiency was excluded, plasma FPS levels were significantly lower in individuals with PSH when compared to those without [72 + 13 vs 91 + 21 UI/dL; p = 1.86 10−6, mean + SD for PSH carriers (n = 21) or controls (n = 1236) respectively]. We provide strong evidence that the rare PSH variant is associated with VT in unselected individuals.

Mutations in BMPR2 are not present in patients with pulmonary hypertension associated with congenital diaphragmatic hernia

BackgroundCongenital diaphragmatic hernia (CDH) is a prevalent major congenital anomaly with significant morbidity and mortality. Thirty to 40% mortality in CDH is largely attributed to pulmonary hypoplasia and pulmonary hypertension (PH). We hypothesized that the underlying genetic risk factors for hereditary PH are shared with CDH associated PH. MethodsParticipants were recruited as part of the Diaphragmatic Hernia Research & Exploration; Advancing Molecular Science (DHREAMS) study, a prospective cohort of neonates with a diaphragmatic defect enrolled from 2005 to 2012. PH affected patients with available DNA for sequencing had one of the following: moderate or severe PH on echocardiography at 3months of age; moderate of severe PH at 1month of age with death occurring prior to the 3month echocardiogram; or on PH medications at 1month of age. We sequenced the coding regions of the hereditary PH genes bone morphogenetic protein receptor type II (BMPR2), caveolin 1 (CAV1) and potassium channel subfamily K, member 3 (KCNK3) to screen for mutations. ResultsThere were 29 CDH patients with PH including 16 males and 13 females. Sequencing of BMPR2, CAV1, and KCNK3 coding regions did not identify any pathogenic variants in these genes. Type of studyPrognosis study Level of evidenceLevel IV

2 &lt;b&gt;．遺伝性プロテインミスフォールディング病に対する創薬 &lt;/b&gt;

2 &lt;b&gt;．遺伝性プロテインミスフォールディング病に対する創薬 &lt;/b&gt;

A Sinister Cause of Bruising in a “Well” Neonate

Neonatal purpura fulminans is a clinical entity characterised by echymotic skin lesions, consumptive coagulopathy and widespread micro and macro vascular thrombosis. This can be hereditary or secondary to acquired causes. Clinical presentation of hereditary Protein C deficiency can be solely limited to localised echymotic skin lesions in the early stage, with wide spread vascular thrombosis ensuing eventually, in the absence of timely Protein C replacement. A case of a neonate presenting with localised scalp ecchymosis at birth, that soon progressed to skin necrosis and renal vein thrombosis is discussed. The report aims to raise awareness among neonatologists regarding the need to consider this rare condition with potentially devastating consequences, while evaluating bruise-like skin lesions in neonates.

Pedigree survey in a family with hereditary protein S deficiency

Objective: To observe the clinical feature of familiar hereditary protein S deficiency(HPSD), and to explore the related gene mutations. Methods: A total of seven family members were enrolled in this study and examined during the June to September 2015. Medical histories of the families were analyzed to detect HPSD according to the diagnostic criteria. PROS1 genes of the proband and her family were analyzed. DNA was extracted from peripheral blood. The 15 exons and their intron-exon boundaries of PROS1 were amplified with PCR, and the PCR products were sequenced and analyzed to identify potential mutations. Medical histories from the family members died prior this study were also obtained. Results: Four out of 7 family members of 2 generations were diagnosed as HPSD. The proband suffered from pulmonary embolism, her elder brother suffered from cerebral infarction and her niece suffered from deep vein thrombosis. A missense mutation at the 1063 bp of cDNA(c.1063C>T)was detected in the exon 10 of PROS1, which resulted in arginine 355 to cysteine replacement in the first ball domain of laminin of the protein S(p.R355C). Conclusion: HPSD is an autosomal dominant genetic disease, patients often suffer from recurring vein thrombosis and pulmonary embolism. A missense mutation(c.1063C>T, p. R355C)of PROS1 was discovered in this Chinese family with HPSD, thus, this mutation might be the genetic basis responsible for these family members with HPSD .

Reep1 null mice reveal a converging role for hereditary spastic paraplegia proteins in lipid droplet regulation.

Hereditary spastic paraplegias (HSPs; SPG1-76 plus others) are length-dependent disorders affecting long corticospinal axons, and the most common autosomal dominant forms are caused by mutations in genes that encode the spastin (SPG4), atlastin-1 (SPG3A) and REEP1 (SPG31) proteins. These proteins bind one another and shape the tubular endoplasmic reticulum (ER) network throughout cells. They also are involved in lipid droplet formation, enlargement, or both in cells, though mechanisms remain unclear. Here we have identified evidence of partial lipoatrophy in Reep1 null mice in addition to prominent spastic paraparesis. Furthermore, Reep1-/- embryonic fibroblasts and neurons in the cerebral cortex both show lipid droplet abnormalities. The apparent partial lipodystrophy in Reep1 null mice, although less severe, is reminiscent of the lipoatrophy phenotype observed in the most common form of autosomal recessive lipodystrophy, Berardinelli-Seip congenital lipodystrophy. Berardinelli-Seip lipodystrophy is caused by autosomal recessive mutations in the BSCL2 gene that encodes an ER protein, seipin, that is also mutated in the autosomal dominant HSP SPG17 (Silver syndrome). Furthermore, REEP1 co-immunoprecipitates with seipin in cells. This strengthens the link between alterations in ER morphogenesis and lipid abnormalities, with important pathogenic implications for the most common forms of HSP.

Roles of transferrin receptors in erythropoiesis.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands.

Hereditary protein C deficiency in a Saudi neonate with bilateral adrenal gland haemorrhages: A rare case report

Abstract This case report describes a full-term 4-day-old Saudi new-born girl diagnosed with hereditary protein C deficiency, who presented with bilateral adrenal gland haemorrhages, intracranial haemorrhage and purpura fulminans. She was born to a consanguineous couple after an unremarkable pregnancy involving a primigravida mother. Her parents were asymptomatic for protein C deficiency. During treatment, her adrenal haemorrhage resolved, but she developed hydrocephalus complicated by cerebral palsy that was initially treated with regular fresh frozen plasma (FFP) and later by low-molecular-weight heparin prophylaxis. Association of protein C deficiency and adrenal haemorrhage in neonates has not been reported previously, and this appears to be the first such case report of its type. Although protein C deficiency may be acquired, the case under consideration proved to be a homozygous hereditary defect. Further genetic studies are required to identify the aetiology of this rare association.

Experimental models of restrictive cardiomyopathy

Restrictive cardiomyopathy (RCMP) is characterized by the isolated diastolic ventricular dysfunction due to increased myocardial stiffness. Animal models of RCMP include the models of acquired and hereditary RCMP as well as the models in large animals. Acquired RCMP is observed in exogenous iron overload, radiation-induced myocardial fibrosis, eosinophilic myocarditis, systemic sclerosis, and amyloidosis. Genetic models of RCMP mimic clinical scenarios of hereditary hemochromatosis and sarcomeric protein mutations. A new approach to modeling RCMP is genetic modification of fruit flies. There are several critical characteristics of successful animal model of RCMP: 1) severe increase in left ventricular end-diastolic pressure in response to volume load; 2) increased ventricular stiffness in the in vitro settings; 3) biatrial enlargement; 4) lack of left ventricular dilation and advanced myocardial hypertrophy.

Hereditary protein S deficiency leads to ischemic stroke.

Hereditary protein S (PS) deficiency is an independent risk factor for venous thromboembolism. However, the correlation between PS and arterial thrombotic disease, such as cerebral thrombosis, is not clear. The present study focused on the molecular mechanisms underlying ischemic stroke caused by a PS gene mutation in one family. The activity of antithrombin, protein C and PS in the plasma of the proband was measured, and the genes encoding PS were amplified and sequenced. The cellular localization and expression of PS were analyzed in HEK-293 cells. The proband was a 50-year-old male. Plasma PS activity of the proband was 38.9%, which was significantly decreased compared with normal levels. Sequencing analysis revealed a PROS1 c.1486_1490delGATTA mutation on exon 12. This frameshift mutation converts Asp496 in the precursor PS into the termination codon. In addition, the PROS1 mutation was correlated with low PS activity in the family. Functional tests revealed that the mutant protein aggregated in the cytoplasm and its secretion and expression decreased. In conclusion, protein S mutation appeared to be the primary cause of thrombosis in the family of the present study. However, the correlation between PS deficiency and ischemic stroke requires further investigation.

Compound heterozygous protein C deficiency in a family with venous thrombosis: Identification and in vitro study of p.Asp297His and p.Val420Leu mutations

Hereditary protein C deficiency (PCD) is an autosomal inherited disorder associated with high risk for venous thromboembolism (VTE). This study aimed to explore the functional consequences of two missense mutations, p.Asp297His and p.Val420Ile, responsible for type I/II PCD and recurrent deep vein thrombosis (DVT) in a Chinese family. The plasma protein C activities (PC:A) of the proband and his sister were reduced to 4% and 5% of normal activity. However, protein C antigen (PC:Ag) concentrations were not equally decreased, with levels of 90.5% and 88.7%, respectively. Two missense mutations p.Asp297His and p.Val420Leu were identified in the protein C gene (PROC). The PC:A and PC:Ag levels in heterozygous state for p.Asp297His were 66% and 64.8%, whereas in heterozygous state for p.Val420Leu, these levels were 67% and 145%, respectively. Wild type (WT) and two mutant PROC cDNA expression plasmids were constructed and transfected into HEK 293T cells. Western blot analysis revealed that both p.Asp297His and p.Val420Leu showed a normal intracellular protein level. The extracellular protein level and specific activity of p.Asp297His were equally reduced to 37.7±4.3% and 22.1±2.5%, respectively. Mutant p.Val420Leu showed a relatively higher PC:Ag level and undetectable PC:A. Immunofluorescence staining revealed that WT and p.Val420Leu proteins were largely co-localized with both the protein disulfide isomerase (PDI) and cis–Golgi Marker (GM130), while the PC p.Asp297His mutant protein was mainly co-localized with PDI and much less co-localized with GM130. The thrombosis symptom in this family was associated with the two missense mutations in the PROC gene.

Prenatal diagnosis of periventricular venous infarction in utero: a case with hereditary protein C deficiency

Abstract A 36-year-old primigravida woman with a normal pregnancy course presented with fetal unilateral focal ventriculomegaly on routine ultrasonography performed at 28 weeks of gestation. Periventricular venous infarction (PVI) in utero was diagnosed with fetal magnetic resonance imaging (MRI). The neonate was born at term uneventfully and in utero PVI was confirmed by MRI after birth. The neonate was diagnosed with hereditary protein C deficiency after coagulation laboratory studies. At 10 months of age, the infant presented with mild retardation of motor development. This is the first report about prenatally diagnosed PVI in utero by fetal MRI. When focal, unilateral enlargement of the ventricles is detected in utero by prenatal ultrasonography, it is important to consider PVI and perform confirmatory fetal MRI.

冠動脈疾患を伴う先天性プロテイン S 欠乏症3例の経験

冠動脈疾患を伴う先天性プロテイン S 欠乏症3例の経験

Sudden sensorineural hearing loss and polymorphisms in iron homeostasis genes: new insights from a case-control study.

Background. Even if various pathophysiological events have been proposed as explanations, the putative cause of sudden hearing loss remains unclear. Objectives. To investigate and to reveal associations (if any) between the main iron-related gene variants and idiopathic sudden sensorineural hearing loss. Study Design. Case-control study. Materials and Methods. A total of 200 sudden sensorineural hearing loss patients (median age 63.65 years; range 10–92) were compared with 400 healthy control subjects. The following genetic variants were investigated: the polymorphism c.−8CG in the promoter of the ferroportin gene (FPN1; SLC40A1), the two isoforms C1 and C2 (p.P570S) of the transferrin protein (TF), the amino acidic substitutions p.H63D and p.C282Y in the hereditary hemochromatosis protein (HFE), and the polymorphism c.–582AG in the promoter of the HEPC gene, which encodes the protein hepcidin (HAMP). Results. The homozygous genotype c.−8GG of the SLC40A1 gene revealed an OR for ISSNHL risk of 4.27 (CI 95%, 2.65–6.89; P = 0.001), being overrepresented among cases. Conclusions. Our study indicates that the homozygous genotype FPN1 −8GG was significantly associated with increased risk of developing sudden hearing loss. These findings suggest new research should be conducted in the field of iron homeostasis in the inner ear.

Hereditary protein C deficiency caused by compound heterozygous mutants in two independent Chinese families

We report two compound heterozygous mutants that caused severe type I protein C (PC) deficiency in two independent Chinese families.PC antigen was determined by enzyme-linked immunosorbent assay (ELISA), and PC activity was measured by chromogenic assay. Genetic mutations were screened with polymerase chain reaction (PCR) followed by direct sequencing. PC mutants were transiently expressed in COS-7 cells for the evaluation of PC secretory activity and function. The subcellular location was visualised by immunofluorescence assay. The structural analysis of mutation was performed as well.Compound heterozygous mutations of Arg178Trp and Asp255His with reduced PC activity and antigen levels were identified in Proband 1, a 28-year-old male with deep vein thrombosis (DVT) and pulmonary embolism. The other mutations of Leu-34Pro and Thr295Ile with reduced PC activity and antigen levels were identified in Proband 2, a 19-year-old male with DVT. The PC activities with Arg178Trp, Asp255His, Leu-34Pro and Thr295Ile mutations decreased significantly. Immunofluorescence assay demonstrated that only trace amount of PC with novel Thr295Ile mutation was transported to the Golgi apparatus. Subsequent structural analysis indicated severe impairments of intracellular folding and secretion.The two rare compound heterozygous mutations could cause type I PC deficiency via impairment of secretory activity of PC.

Abstract 1554: Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression

Abstract Breast cancer is the first cause of death by cancer in women in Chile as in many countries around the world. Until today, BRCA1 and BRCA2 are the most relevant genes for breast cancer high risk. Moreover, different studies have shown that BRCA1 is under expressed in breast tumors. Our group has found that in a high percentage of hereditary tumors BRCA1 protein have a diminished or null expression, or is mislocalized to the cytoplasm (unpublished data). In this regard, it has become a goal for us to identify if mislocalization and/or loss of expression of BRCA1 and BRCA2 could be related to somatic mutations in the tumors. In this work, we sequenced BRCA1 and BRCA2 in DNA extracted from 14 fresh/frozen tumors. DNA was isolated from each tumor and amplified by PCR in 5 multiplexes (a total of 93 amplicons) considering all exons and intron/exon junctions with approximately 30bp of intronic sequence each side. Multiplexes were then pooled for each patient and processed for sequencing in the GS Junior Roche. All reads were processed with AVA software to detect nucleotide changes in BRCA1 and BRCA2 sequences and analyzed manually to discard misinterpretations. All mutations identified were confirmed by Sanger. From the same tumors we obtained a second section that was formalin fixed and paraffin embedded. This FFPE fragment was cut in 4 μm sections and analyzed by immunohistochemistry and immunofluorescence to assess BRCA1 expression and localization. We found a total of 5 mutations, 3 in BRCA1 and 2 in BRCA2, in 6 tumors. Interestingly, one BRCA1 mutation present in 7 to 20% of reads, was found in 5 tumors. Among those, two tumors presented two different mutations in BRCA2, in addition. The last tumor presented one extra mutation in BRCA1. BRCA1 expression analysis was coherent between immunohistochemistry and immunofluorescence assays. In general 9 tumors showed weak or negative BRCA1 nuclear expression and 5 mainly moderate nuclear staining. In addition 6 tumors presented moderate to strong cytoplasmic expression of the protein, considered abnormal. Considering tumors with weak or negative expression of BRCA1, 40% presents BRCA1 or BRCA2 mutations. The relevance of the absence of BRCA1 expression, or function impairment of BRCA1 and/or BRCA2 due to somatic mutations, relays on the possible treatment with PARP1 inhibitors. Supported by FONDECYT 1120200. Citation Format: Carolina Alvarez, David Wiener, Patricia Gajardo, Wanda Fernandez, Valeria Cornejo, Jorge Gamboa, Pilar Carvallo. Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1554. doi:10.1158/1538-7445.AM2014-1554

Hereditary transthyretin amyloidosis

Hereditary amyloidosis is an autosomal dominant fatal multisystem disease caused by extracellular deposition of misfolded proteins and, therefore represents a hereditary protein folding or deposition disease that leads to progressive organ damage and eventually death. In most instances mutations within the transthyretin gene are the underlying cause. The main manifestation is a rapidly progressing axonal sensorimotor and autonomic polyneuropathy (familial amyloid polyneuropathy, FAP). Cardiac involvement is frequent in FAP and additional manifestations include the gastrointestinal tract and the eyes. A second manifestation type is cardiomyopathy with little or no polyneuropathy (familial amyloid cardiomyopathy, FAC). For therapy, orthotopic liver transplantation has been established for 25 years. Recently, the oral agent tafamidis, a transthyretin stabilizer, was licensed for treatment of stage 1 polyneuropathy. Additional treatment options are currently being studied.

The genomic architecture of the PROS1 gene underlying large tandem duplication mutation that causes thrombophilia from hereditary protein S deficiency.

Hereditary protein S deficiency from a mutation in the PROS1 gene causes a genetic predisposition to develop venous thromboembolic disorders in humans. Recently, the acknowledgment of the clinical significance of large copy number mutations in protein S deficiency has increased. In this study, the authors investigated the genomic architecture of PROS1 in order to understand the microscopic sequence environment leading to large intragenic copy number mutations in the gene. The study subjects were 3 unrelated male patients with hereditary protein S deficiency from a tandem duplication mutation involving exons 5-10 of PROS1. Breakpoint analyses revealed 10-bp microhomology sequences in the intervening sequence (IVS)-4 and IVS-10 at the duplication junction without additional sequence changes, suggesting a single replication-based event as the potential molecular mechanism of rearrangement and founder effect in the mutant alleles. Further analyses on nucleotide sequences flanking the microhomology sequence revealed the presence of a repeat element (LTR-ERV1) and quadruplex-forming G-rich sequences in IVS-4. The results from genotyping multi-allelic short tandem repeats supported founder effect in the identical mutations in the 3 unrelated patients. In conclusion, we identified unique genomic architectures in the intervening sequences of PROS1 that underlie a large intragenic tandem duplication mutation leading to inherited thrombophilia.

Hereditary protein S deficiency presenting acute pulmonary embolism

Protein S deficiency is one of the several risk factors for thrombophilia and can cause blood clotting disorders such as deep vein thrombosis and pulmonary embolism. A 54-year-old man was admitted with the complaint of dyspnea and was diagnosed with pulmonary embolism. The patient had very low level of free protein S, total protein S antigen, and protein S activity (type I protein S deficiency). In history taking, we found that his mother, 78 year old, had a history of same disease 10 years ago, and confirmed the pronounced low level of protein S. The patient’s son also had very low level of protein S, however there had not been any history of pulmonary embolism yet. This case study suggests that asymptomatic persons with a family history of protein S deficiency and pulmonary embolism should be checked regularly for early detection of the disease, as protein S deficiency can be suspected.