Laboratory Diagnosis of Congenital Factor V Deficiency, Routine, Specific Coagulation Tests with Molecular Methods

Erratum: The correct affiliation of corresponding author of this manuscript has been edited as follows:"Akbar Dorgalaleh: Department of Hematology and Blood Transfusion, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran."Factor V (FV) deficiency is a rare bleeding disorder (RBD) that inherit in autosomal recessive manner. Diagnosis of FV deficiency (FVD) is made by routine coagulation tests, FV activity and molecular analysis. In patients with FVD, routine coagulation tests including activated partial thromboplastin time (APTT), prothrombin time (PT) and evenbleeding time (BT) are prolongedwhile thrombin time (TT) is normal. FV activity assay can use for confirmation of diagnosis as well as for differential diagnosis with acquired forms of disease. Mixing study can be used for screening of inhibitor against FV. In this situation, addition of normal plasma cannot correct prolonged PT and PTT while in congenital FVD prolongation is corrected. Molecular diagnosis of FVD is straightforward but due to large size of FV gene and genetic variability molecular diagnosis is restricted to research laboratory

Burden of Congenital Factor XIII Deficiency in Iran

Congenital factor XIII (FXIII) deficiency is a rare coagulopathy with the highest incidence in Iran. Iranian patients with FXIII deficiency (FXIIID) presented high rate of bleeding episodes, some of them are major cause of disability and mortality among these patients. Hemarthrosis and intracranial hemorrhage (ICH) can affect activity and social productivity of patients. ICH, recurrent miscarriage and umbilical cord bleeding are the major cause of mortality. Hematoma, and prolonged menstrual bleeding as well as post-surgical bleeding are other significant bleeding in Iranian patients with FXIIID. Present of severe life threatening bleeding episodes and other notable bleedings, can significantly reduce working activities and social productivities of patients. Although Iranian patients with FXIIID, experienced significant diseases related complications, early diagnosis accompany by appropriate therapeutic regimes can prevent most of these problems

Identification of the First Iranian Family with “γArg275Cys” Mutation (Fibrinogen Tokyo II)

Background: Inherited fibrinogen deficiencies areclassified into two categories: quantitative, including afibrinogenemia and hypofibrinogenemia and qualitative, including dysfibrinogenemia. Any mutation in fibrinogen genes accounts for one of these disorders.Case report: This article reports an Iranian family with dysfibrinogenemia without any clinical signs accidentally diagnosed by routine coagulation tests with slightly elevated PT and APTT a few years ago. Fordeterminationof disease causing genetic aberration in fibrinogen genes, DNA sequencing of three hot spots of these genes (i.e. exon 2 of FGA,exon 2 of FGB and exon 8 of FGG)was performed. Analysis of sequencing results revealed a heterozygous missense mutation c.901 C>T (Arg275Cys) in exon 8 of FGG in mother and children.No mutationwas detected in father’s sample.Fibrinogen with this mutation is known as Tokyo II.Conclusion: γArg275Cys is a heterozygous mutation that impairs the function of fibrinogen andhas been solely reported in dysfibrinogenemic patients. Clinical findings in this family (no history of bleeding and thrombosis) were compatible with molecular results, because fibrinogen Tokyo II does not have a thrombotic or hemorrhagic nature and lack of clinical signs in this family is not unexpected.

Bleeding Episodes Among Patients with Congenital Fibrinogen Disorders, a Study On 12 New Iranian Patients

Background: Congenital fibrinogen disorders (CFDs) comprise about 10% of rare bleeding disorders (RBDs). CFDs are divided into two groups of quantitative (afibrinogenemia and hypofibrinogenemia) with autosomal recessive inheritance pattern, and qualitative (dysfibrinogenemia, hypodysfibrogenemia) disorders, mainly with autosomal dominant inheritance pattern. Sistan and Baluchestan Province in Iran, with its high rate of consanguineous marriages, has a high incidence of RBDs including CFD. In the current study, we report clinical manifestations of patients with CFDs.Methods: Twelve new Iranian patients from Sistan and Baluchestan Province with different types of CFDs were selected for this study. Diagnosis of CFDs was based on clinical features and familial history followed by laboratory assessment by routine and specific coagulation tests including prothrombin time (PT) and activated partial time tests (APTT), as well as FI activity assay by Clauss method.Results: Out of 12 patients, 3(25%) had afibrinogenemia, 7(58.3%) had hypofibrinogenemia while 2(16/7%) were suspected of having dysfibrinogenemia. Although umbilical cord bleeding (UCB) 9(75%) was the most common clinical presentation among the study population, this feature was not observed among patients with dysfibrinogenemia. Hematoma (100%) was the most common presentation of patients with dysfibrinogenemia.  Conclusion: Results of this study revealed that some clinical presentations are the diagnostic features of CFDs and can be used for precise and in-time diagnosis CFDs in conjunction with family history and laboratory findings.Keywords: Fibrinogen Deficiency; Congenital Afibrinogenemia; Blood Coagulation Disorder; Afibrinogenemi

Complete Genotype and Clinical Phenotype of Hemophilia B: A Study on Iranian Patients

Background: Hemophilia B which refers to the deficiency or functional defect of factor IX (FIX) is typically an X-linked bleeding condition that arises from heterogeneous mutations of the FIX gene (F9). The number of hemophilia cases in Iran is considerable and currently, about 1118 Iranian patients are suffering from hemophilia B, although a small number of them underwent genetic investigations. Here we assessed molecular defects and also laboratory and clinical findings of 10 Iranian cases with hemophilia B. Materials and Methods: A total of 10 cases with hemophilia B were enrolled in the study. Patients were clinically examined by a hematologist and their previous medical documents were surveyed carefully. Routine coagulation tests and FIX activity and antigen assays were performed for the studied patients. Genotyping of F9 for identifying genetic mutations was conducted by the Sanger sequencing method following PCR amplification of the promoter region and all the eight exons of the F9 gene. Results: The mean age of patients was 4 years (9 months to 16 years) and consanguinity was reported in 80% of cases. Patients were commonly manifested by hematoma (90%), epistaxis (80%), and hemarthrosis (70%) and the severity of the disorder was severe (70%) or moderate (30%). In nine out of 10 patients a genetic defect in F9 gene we detected including three missense (c.304T>C, c.1007T>A, c.191G>A) and three nonsense mutations (c.892C>T, c.880C>T, c.1113C>A). Based on the FIX variant database (http://www.factorix.org), five mutations have been reported previously, but mutation c.1007T>A (p.Ile336Asn) seems to be a novel mutation. Conclusion: Our results indicated the heterogeneous molecular defects of hemophilia B in Iran, as recorded in the FIX mutation database. Moreover, no specific genotype-phenotype association was observed in studied subjects

Coagulation Factor XIII-A A614T gene Variation is Suggestive of Founder Effect in Iranian Patients with Severe Congenital Factor XIII Deficiency

Background: Factor XIII (FXIII) is a heterotetramer consisting of two subunits, FXIII-A and FXIII-B. Several common gene variations were observed in the FXIII-A gene with an obvious ethnic difference. This study assessed the prevalence of A614T as a common FXIII-A gene variation among Iranian patients with FXIII deficiency (FXIIID). Materials and Methods: This study was conducted on eighty Iranian unrelated individuals with FXIIID. Genotype analysis for FXIII-A A614T gene variation was performed for all individuals. Results: Molecular analysis of these Iranian populations revealed that all studied patients were homozygous for the T allele at codon 204 of the FXIII-A1 subunit. Conclusion: Present of T allele at codon 204 of FXIII-A1 subunit among all study population can be suggestive of founder effect. &nbsp

Prevalence of Rare and Common Bleeding Disorders in Kurdistan Province of Iran

Background: Congenital bleeding disorders (CBD) are a group of coagulopathies with different clinical and laboratory features. The prevalence of these disorders in different parts of the world is variable. Iran as a country with a high rate of parental consanguinity has a high rate of CBDs. This study was to report the prevalence of these disorders in Kurdistan province, west of Iran.Methods and materials: This descriptive study was conducted on patients suspected of a congenital bleeding disorder referred to hemophilia center of this province for evaluation of underlying bleeding diathesis. Diagnosis and classification of disorders were made by routine and specific laboratory tests.Results: Out of 107 patients, 65.4% affected by common bleeding disorders (hemophilia A and B), 23.4% affected by rare bleeding disorders (RBDs) and 11.2% had inherited platelet disorders. Factor VII deficiency (64%) was the most common RBDs and 9 patients had von Willebrand disease. Out of three patients with inherited platelet disorders, two had Glanzmann thrombasthenia.Conclusion: CBD pattern though has similar patterns with total pattern of the country, some of the inherited platelet disorders are more common in Kurdish province. Determination of prevalence and distribution of these disorders can improve health system planning and resource allocation.Keywords: Congenital bleeding disorders, Rare bleeding disorders, Common bleeding disorders, inherited platelet disorder

Thrombin Activatable Fibrinolysis inhibitor Thr 325 Ile polymorphism in fetuses with factor XIII deficient family history and Intracranial hemorrhage

Background: Factor XIII Deficiency (FXIIID) is an inherited rare bleeding disorder with some life threatening clinical manifestation including Intracranial Haemorrhage (ICH). Among all polymorphisms found in FXIIID, Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Thr325Ile gene polymorphism increases probability of ICH about 20 fold in patients with FXIII .So, in this study we aimed to evaluate TAFI Thr 325 Ile polymorphism in Chorionic villus samples (CVS) of fetuses with positive family history of FXIIID and ICH.Materials and Methods: This study was performed on chorionic villus of pregnant mothers ´ with positive history of FXIIID accompanied with ICH in first-degree relatives of their fetus. All parents of the fetuses were completed consent form for doing Prenatal diagnosis (PND). Chorionic villus DNA was extracted from each sample using the DNA extraction kit and PCR-RFLP was performed for TAFI Thr 325Ile polymorphism in Exon 4 of FXIII A gene.Results: All of 8 fetuses had positive family history of FXIIID. Seven out of eight fetuses (87.5%) had a family member with CNS bleeding due to FXIIID. Four fetuses had history of death due to FXIIID. There were 5 case (62.5%) that were homozygote for TAFI Thr 325 Ile, one (12.5%) was heterozygote and two (25%) were non mutant. Conclusion: Detection of TAFI Thr 325 Ile polymorphism by PND program in fetuses with positive family history of ICH is seems necessary and it will help to fill many gaps in preventing life threatening features of FXIIID in newborn at the time of delivery by prophilaxy receiving and precautionary measures

Congenital Prothrombin Deficiency

Congenital prothrombin deficiency is an extremely rare hemorrhagic disorder with estimated prevalence of 1 per 2,000,000 in the general population. Since the disorder is an autosomal recessive disorder, the disorder is more frequent in areas with high rate of consanguinity. Clinical manifestations of disorder are highly variable ranging from mild bleeding episodes to severe life-threatening hemorrhages. The disorder can be diagnosed based on routine and specific tests. No specific factor II concentrate is available, but patients can receive fresh frozen plasma and prothrombin complex concentrate (PCC). Traditionally patients with prothrombin deficiency receive on-demand therapy, but secondary prophylaxis can be used for those patients with high risk of severe life-threatening bleeding. With timely diagnosis and appropriate management of disorder, the quality of life in these patients can significantly improve.   Keywords: Prothrombin deficiency, Clinical manifestations, Diagnosis, Treatmen

c.559 T>C as The Most Common Mutation of Factor XIII Deficiency in Iranian Patients is not Restricted to Southeast Iran

Background: Iran has a large group of patients with severe congenital factor XIII deficiency (FXIIID) and Trp187Arg mutation that is most disease causing mutation of FXIII in the world is only observed in southeast of Iran with 352 patients with FXIIID. 743 patients with FXIIID was observed in 17 provinces of Iran but Tehran city with more than 12 million population has no any registered patient with FXIIID. Here we described first case with severe congenital FXIIID in Tehran Province with underline FXIII mutation.Methods: A neonate with prolonged umbilical cord bleeding was referred to hemophilia center. Patient was screened by routine coagulation tests and by clot solubility test. After observation of normal routine tests and abnormal clot solubility patient was underwent a full sequencing of FXIII-A gene. For confirmation of detected mutation in FXIII-A gene, exon 4 was amplified by PCR and cleaved by Eco130I restriction enzyme.Results: We found first case with severe congenital FXIIID in Tehran Province with Trp187Arg mutation in exon 4 of FXIII-A gene. Patient’s parents were heterozygote for this mutation.Conclusion: Trp187Arg mutation of FXIII-A is the most common mutation in Iranian patients with FXIIID and is not restricted to southeast of Iran.Keywords: Factor XIII deficiency, Trp187Arg mutation, Tehran Provinc