Severe Factor V Deficiency Caused by a Novel Compound Heterozygous Mutation: Factor V G1617V and 1-Bp Insertion

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2268-2268
Author(s):  
Keiko Shinozawa ◽  
Yuri Okimoto ◽  
Kagehiro Amano ◽  
Harumi Kakuda ◽  
Takeshi Hagiwara ◽  
...  
Keyword(s):  
Healthy Subjects ◽  
Family Members ◽  
Compound Heterozygous Mutation ◽  
Heterozygous Mutation ◽  
Severe Bleeding ◽  
Compound Heterozygous ◽  
Factor V ◽  
Bleeding Tendency ◽  
Wild Type ◽  
Rt Pcr

Abstract Abstract 2268 Introduction: Congenital factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. There is poor correlation between FV activity in plasma and symptoms of bleeding tendency. In the present study, we identified a novel compound heterozygous mutation in the FV gene (F5) in a Japanese patient with severe bleeding symptoms. Patient and methods: The patient was a young boy in his teens. He suffered from intracranial hemorrhage after birth and he also experienced joints and muscle hemorrhage, afterwards. His plasma FV activity was less than 1% and FV antigen was 2%. Sequence analysis of F5, FV protein analysis, and recombinant mutant protein expression experiments were performed. And to evaluate the relation between bleeding symptoms and FV phenotypes, we analyzed the FV level of platelets in the patient and his family members. Our present and previous results were verified. Results: Sequence analysis of F5 in this patient revealed a novel compound heterozygous mutation, G1617V missense mutation in exon 14 and a 1-bp insertion in exon 16 (p.G1645V and c.5255–5259 ins A). The patient's father had a G1617V mutation and his mother had a 1-bp insertion for the heterozygote state, respectively. The patient's two sisters each had also a 1-bp insertion for the heterozygote state. We analyzed the expression of platelet F5 mRNA by semi-quantitative RT-PCR, which showed that platelet F5 mRNA from the patient and his family members were equal to the amount in healthy subjects. Platelet FV protein from family members examined by western blotting detected equal to those of healthy subjects, although the FV bands from the patient were detected only weakly. Platelet FV antigen and activity were measured with an ELISA and a functional assay based on prothrombin time. The patient's platelet FV activity was 1.6% and antigen was 1%, compared with 100% for those of normal subjects. Platelet FV activity and antigen of the patient's father, mother, elder sister and younger sister were 26% and 37.1%, 39.6% and 51.0%, 23.3% and 38.0%, and 35.4% and 56.6%, respectively. In the expression study of pMT2/FV-wild-type and FV-G1617V mutant recombinant proteins in HEK293 cells, the FV antigen levels produced by the FV-G1617V mutant in cell lysates was approximately 73% of wild-type. In conditioned media of the study, FV antigen and specific activity were reduced to approximately 4% and 6% of wild-type, respectively. Discussions: Although there was similar intracellular synthesis of the FV-G1617V mutant and wild-type FV, the results in the conditioned media suggested that secretion of the FV-G1617V mutant was impaired. Platelet FV protein was detected very low by Western blot and ELISA, though platelet F5 mRNA was confirmed to be normal by RT-PCR. Our previous results raise the possibility that in the case of severe FV deficiency, such as that with a 1 % or less plasma FV level, the amount of FV in platelets is important to cope with local bleeding. This patient's platelet FV activity and antigen levels were the lowest of those of a number of FV-deficient patients whom we have analyzed so far, and this patient had severe bleeding symptoms. Conclusion: Our previous studies support the concept that the severity of bleeding is related to FV protein in platelets. Taken together, we believe the severity of this patient's bleeding tendency is caused by a reduction in both platelet and plasma FV, especially by the highly significant decrease in platelet FV. Disclosures: No relevant conflicts of interest to declare.

Novel Compound Heterozygous Factor V Deficiency with Severe Clinical Phenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3490-3490
Author(s):  
Kimberley Talbot ◽  
Jina Song ◽  
Lily Eghdami ◽  
Jeff Hewitt ◽  
Cedric J. Carter ◽  
...  
Keyword(s):  
Protein Folding ◽  
Sequence Analysis ◽  
Dna Sequence ◽  
Metal Ion ◽  
Conflicts Of Interest ◽  
Dna Sequence Analysis ◽  
Severe Bleeding ◽  
Compound Heterozygous ◽  
Factor V ◽  
Bleeding Tendency

Abstract Abstract 3490 Poster Board III-427 Introduction Congenital factor V (FV) deficiency is a rare clotting disorder associated with mild to severe hemorrhagic symptoms and a prevalence of approximately 1 per million in the general population. Patients with FV deficiency normally show very low or unmeasurable plasma levels of functional and immunoreactive FV and are usually homozygous or compound heterozygous for mutations located in the FV gene. Heterozygous carriers have approximately half-normal levels of FV and are usually asymptomatic. Patient History In this study, a proposita now aged 71 is described, who has less than 3% FV activity and is clinically diagnosed as having severe FV deficiency. Since childhood she has exhibited severe bleeding tendency with surgery/dental extraction or trauma. Other symptoms include frequent nose bleeds, bruising easily and profound menorrhagia that led to eventual hysterectomy in her mid-thirties. Parents were clinically asymptomatic suggesting compound heterozygous FV deficiency for this individual. Her children, a son and a daughter, are also asymptomatic and have FV levels of approximately 50-60%. Methods DNA was isolated from peripheral blood leucocytes and the FV exon and flanking intron sequences were amplified by PCR, and subjected to automatic DNA sequence analysis. FV levels were assayed in plasma using conventional clotting assays as well as immunoassays using monoclonal and polyclonal antibodies detecting several regions of FV. Results DNA sequence analysis revealed two mutations: the first in exon 17 (C > T) changed the codon for Leu-1821 to Ser (L1821S), the second in exon 25 (T > G) changed Gly-2192 to Cys (G2192C). Plasma clotting assays (n=2) showed FV activity levels of 0.5±0.015% for prothrombin time and 2.0±0.10% for activated partial thromboplastin time compared to normal pooled plasma. Western blot analysis using a polyclonal antibody demonstrated the patient FV banding pattern was comparable to normal plasma. Densitometric analysis of the specific bands showed that the patient had 9% of the FV antigen level compared to normal pooled plasma. Conclusions Two novel FV mutations have been identified. It has previously been reported in the literature that mutations involving thiols can have deleterious effects on protein folding and secretion. Consequently, the G2192C mutation could alter the FV protein folding and/or secretion which may explain the reduced level of FV antigen detected by immunoassay. In addition, the L1821S mutation is close to a putative activated FV metal ion binding site and may have an important effect on inter subunit reactions and subsequently FV function. Since antigen and activity are discordant, both FV function and secretion appear to be affected by these mutations. Future studies aim to introduce these unique mutations into recombinant FV protein to gain new insight into FV secretion, activity and function. Disclosures: No relevant conflicts of interest to declare.

Identification of a Novel Coagulation Factor X Compound Heterozygous Mutation Associated with Differential Initiating Clotting Pathway Function.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1129-1129
Author(s):  
Amanda L Vanden Hoek ◽  
Kimberley Talbot ◽  
Isis Carter ◽  
Linda Vickars ◽  
Cedric John Carter ◽  
...  
Keyword(s):  
Western Blot ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Compound Heterozygous Mutation ◽  
Heterozygous Mutation ◽  
Severe Bleeding ◽  
Compound Heterozygous ◽  
Bleeding Diathesis ◽  
Intrinsic Pathway ◽  
Extrinsic Pathway

Abstract Abstract 1129 Introduction: Factor × (FX) deficiency is a rare form of haemophilia characterized by a decrease in circulating FX antigen and/or activity levels, which can result in a variable bleeding diathesis. In heterozygous patients, bleeding may be absent or mild while homozygous and doubly heterozygous patients have phenotypes that are often associated with moderate or severe bleeding. Patient History: In this study, a propositus now aged 75 with a moderate bleeding diathesis is described. Neither parent had a history of bleeding. The patient was originally diagnosed as FX-deficient based on clinical measurements of coagulation factors. With prostate surgery, he had unexpected bleeding, that could not be explained surgically, requiring large volumes of plasma and red cell concentrates. Other surgical challenges, including dental extractions, were not complicated by bleeding but were preceded by plasma infusion. Methods: Plasma FX antigen levels were assayed by western blot using FX-specific monoclonal antibodies. To follow activity, prothrombin time and activated partial thromboplastin time clotting tests were used to evaluate the extrinsic and intrinsic initiating branches of coagulation, respectively. The entire F10 gene (8 exons and flanking intronic sequences) was amplified using PCR and sequenced to identify mutations. Results: DNA sequence analysis identified two mutations, which were presumably on different alleles based on a lack of parental bleeding. The first was a previously reported mutation that disrupts the splice site between exons 1 and 2 (IVS1 +1 G>A) and was hypothesized to lead to premature degradation of FX mRNA transcripts (Wang WB et al, Haemophilia 2005). This explains a 50% loss of antigen in our heterozygous patient. The second was a novel mutation at nucleotide 28145 (C>T) which results in an Arg386 to Cys (Arg386Cys) substitution in the serine protease domain of FX. Quantification of plasma FX antigen by western blot analysis revealed 15% of normal, which correlated precisely with 15% extrinsic pathway activity. However, intrinsic pathway clotting activity was only 5% of normal. The fragmentation of FX antigen in plasma after initiation of coagulation was followed over time. When initiated through the extrinsic pathway, the patient's FX fragmentation profile was identical to normal plasma. However, when clotting was triggered through the intrinsic pathway, activation to FXa and appearance of other fragments was notably slower. This further confirms that the patient's novel FX defect predominantly affects the intrinsic pathway while maintaining normal function in the extrinsic pathway. Conclusions: Here we describe a compound heterozygous FX deficiency. The first mutation has been reported once before (IVS1 +1 G>A) and accounts for 50% loss of FX antigen. The second FX mutation we identified is novel and may result in alternate disulfide bond formation; in particular at the nearby sole covalent link between the heavy and light chains of FX. This may explain the 35% further reduction in FX plasma antigen to 15% for this patient. Interestingly, the differential effect of Arg386Cys on the extrinsic and intrinsic coagulation pathways suggests that Arg386 may be involved in the substrate recognition by the intrinsic FIXa/FVIIIa Xase complex. As this Xase functions to amplify coagulation, Arg386Cys may be predicted to most affect hemostasis under severe conditions such as surgery. Production of recombinant FX containing this mutation is currently underway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel mutation in the CHST14 gene causing musculocontractural type of Ehlers-Danlos syndrome

2018 ◽  
pp. bcr-2018-226165 ◽  
Author(s):  
Sapna Sandal ◽  
Anupriya Kaur ◽  
Inusha Panigrahi
Keyword(s):  
Connective Tissue ◽  
Novel Mutation ◽  
Connective Tissue Disorder ◽  
Rare Disorder ◽  
Compound Heterozygous Mutation ◽  
Heterozygous Mutation ◽  
Compound Heterozygous ◽  
Bleeding Tendency ◽  
Ehlers Danlos Syndrome ◽  
Danlos Syndrome

Musculocontractural type of Ehlers-Danlos syndrome (MC-EDS) is a recently recognised connective tissue disorder. MC-EDS is caused by homozygous or compound heterozygous mutation in the carbohydrate sulfotransferase 14 (CHST14) gene on chromosome 15q15. Herein, we report a case of a 3-year-old boy with MC-EDS in whom a novel mutation in the CHST14 gene was discovered. Besides being the second report of this rare disorder from India, the child till 3 years has not had any bleeding tendency as described in the earlier reports of this disorder.

Identification and Cloning of Novel Mutations In a Compound Heterozygous Factor V Deficient Patient

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2210-2210
Author(s):  
Kimberley Talbot ◽  
Jina Song ◽  
Lily Eghdami ◽  
Jessica Tamura-Wells ◽  
Jeff Hewitt ◽  
...  
Keyword(s):  
Western Blot ◽  
Banding Pattern ◽  
Blot Analysis ◽  
Severe Bleeding ◽  
Compound Heterozygous ◽  
Factor V ◽  
Bleeding Tendency ◽  
Novel Mutations ◽  
Deficient Patient ◽  
Pre Treatment

Abstract Abstract 2210 Introduction: Congenital factor V (FV) deficiency is a rare clotting disorder associated with mild to severe hemorrhagic symptoms and a prevalence of approximately 1 per million in the general population. Patients normally present with very low or unmeasurable levels of functional and/or immunoreactive FV and are usually homozygous or compound heterozygous for mutations located in the F5 gene. Heterozygotes typically have approximately half-normal levels of FV and are asymptomatic, whilst compound heterozygotes may show mild to severe bleeding diathesis. Patient Description: A proband now aged 73, was clinically diagnosed since childhood as having severe FV deficiency (<3% FV activity by clinical lab analysis), exhibiting severe bleeding tendency with surgery or dental extraction. Other symptoms included frequent nose bleeds, bruising easily and profound menorrhagia that led to eventual hysterectomy in mid-thirties. Additional routine clinical assays for clotting factors were normal. There was no history of parental bleeding tendency. Methods: DNA was isolated from peripheral blood leucocytes. Exonic and flanking intronic sequences of F5 were amplified by PCR and subjected to automated nucleotide sequencing. To investigate the role of identified mutations in FV function and protein secretion, wildtype (WT) FV and FV variants consistent with mutations identified for the patient using the QuikChange Site-Directed Mutagenesis Kit were cloned into the pED-FV-1033 expression vector. This produced recombinant single chain FV that requires proteolytic activation to express cofactor function. Plasmids encoding WT FV, and the mutation identified in the patient were transfected into baby hamster kidney cells (BHK) using Lipofectamine and stable clones were established. Secretion of FV protein was quantified by commercial enzyme immunoassay (EIA), and FV activity was evaluated using conventional prothrombin time (PT) and activated partial thromboplastin time (APTT) clotting assays. Western blot analysis using monoclonal antibodies to both FV heavy (FVaH) and light chains (FVaL) detected FV antigen and fragmentation profiles. Results: Based on the lack of parental bleeding, compound heterozygosity was probable for this FV deficient patient because the DNA sequence analysis revealed two novel mutations; the first changed the codon for Leu1821 to Ser (L1821S), the second changed Gly2192 to Cys (G2192C). Clotting assays showed low patient plasma FV activity of 0.5±0.015% for PT and 2.0±0.10% for APTT compared to normal pooled plasma. Western blot analysis demonstrated the patient FV banding pattern was comparable to normal plasma, whilst densitometric analysis of the specific bands showed that the patient had 9% of the normal FV antigen level. Recombinant FV secretion detected by EIA from various clones ranged from 0 to 16.6ng/mL. L1821S (28%) and G2192C (25%) mutants secreted lower FV protein compared to WT. Clotting assays (n=2) showed that Leu1821S activity levels were 15% that of WT in contrast to G2192C where no FV clotting activity was detectable. Compared to WT, L1821S had 55% and G2192C had only 1.4% of the specific activity.Western blot analysis demonstrated the L1821S banding pattern was comparable to WT whilst G2192C lacked several fragments. Prolonged thrombin (FIIa) pretreatment of FV clones confirmed WT and both mutants were cleaved to FVaL (∼74k Da) and FVaH (∼105 kDa). In addition, FIIa pre-treatment enhanced clotting activity for most of the clones evaluated. Interestingly, the inactive G2192C mutant acquired modest FV function after pre-incubation with FIIa. Conclusions: Two novel FV mutations that affect both function and secretion have been identified in a FV deficient patient and have been cloned. The L1821S mutation is located close to a FV metal ion binding site that may affect function. G2192C introduces a thiol into FV, reported in the literature to have deleterious effects on protein folding, which may explain attenuated secretion and lack of clotting activity. Only after prolonged pre-treatment with FIIa, G2192C function was acquired, which may suggest that this mutation stabilizes the pro-cofactor conformation of FV. Low in vitro secretion of mutant FV compared to WT corresponds to low patient plasma FV antigen, which is compounded by profound loss of function in G2192C. Further biochemical studies are underway to understand the effect of G2192C on FV activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel Compound Heterozygous Mutation in ABCB4 Gene Leading to Cholelithiasis, Progressive Familial Intrahepatic Cholestasis (Type 3), and Cirrhosis in a Child

2020 ◽  
Vol 10 (01) ◽  
pp. e134-e136
Author(s):  
Nida Mirza ◽  
Smita Malhotra ◽  
Anupam Sibal
Keyword(s):  
Intrahepatic Cholestasis ◽  
Gall Stone ◽  
Compound Heterozygous Mutation ◽  
Heterozygous Mutation ◽  
Compound Heterozygous ◽  
Initial Symptom ◽  
Progressive Familial Intrahepatic Cholestasis ◽  
Presenting Symptoms ◽  
Abcb4 Gene ◽  
Type 3

AbstractProgressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal recessive disorders of childhood which presents with intermittent or progressive episodes of cholestasis, with jaundice and pruritus as most common presenting symptoms. PFIC type 3 occurs due to mutations in the ABCB4 gene, mutation in this gene has wide spectrum of features which include intrahepatic stones, cholelithiasis, PFIC type 3, and intrahepatic cholestasis of pregnancy. Here, we are reporting a peculiar case of young male adolescent with novel variant compound heterozygote missense mutation in ABCB4 gene who had gall stone as initial symptom, followed by symptoms of PFIC and eventually decompensated chronic liver disease.

scholarly journals Leptin Receptor Compound Heterozygosity in Humans and Animal Models

2021 ◽  
Vol 22 (9) ◽  
pp. 4475
Author(s):  
Claudia Berger ◽  
Nora Klöting
Keyword(s):  
Food Intake ◽  
Animal Models ◽  
Leptin Receptor ◽  
Compound Heterozygous Mutation ◽  
Heterozygous Mutation ◽  
Compound Heterozygous ◽  
Compound Heterozygosity ◽  
Therapy Options ◽  
Obese Phenotype ◽  
Deficient Mice

Leptin and its receptor are essential for regulating food intake, energy expenditure, glucose homeostasis and fertility. Mutations within leptin or the leptin receptor cause early-onset obesity and hyperphagia, as described in human and animal models. The effect of both heterozygous and homozygous variants is much more investigated than compound heterozygous ones. Recently, we discovered a spontaneous compound heterozygous mutation within the leptin receptor, resulting in a considerably more obese phenotype than described for the homozygous leptin receptor deficient mice. Accordingly, we focus on compound heterozygous mutations of the leptin receptor and their effects on health, as well as possible therapy options in human and animal models in this review.

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