Study on the molecular mechanisms of a novel large deletion of FXIIIA mRNA in a new hereditary factor XIII deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the mechanisms of DelCD11-279 of factor XIII subunit A mRNA in the pathogenesis of hereditary factor XIII deficiency.</p><p><b>METHODS</b>The recombinant plasmids containing pET-22b(+)/FXIIIA of normal subject and proband's mother and pET-22b(+)/FXIIIA-Del of the proband were constructed and transformed into E. coli BL21. Expressing protein was analyzed by the SDS-PAGE and purified by Ni-NTA resin. Purified proteins were detected by the Western-blot. The activity of purified protein was detected by the incorporation test with EZ-LinkTM5-(Biotinamido) Pentylamine.</p><p><b>RESULTS</b>The recombinant plasmids containing pET-22b(+)/FXIIIA and pET-22b(+)/FXIIIA-Del which constructed and identified successfully by enzyme digestion and PCR, were transformed into E. coli BL21 and efficiently expressed by IPTG induction. The molecular weights of expressing proteins are 83 200 and 51 900 by the SDS-PAGE. Expressing proteins were purified by Ni-NTA resin, and were proved to be human FXIIIA proteins by Western-blot. Purified protein activity of proband's mother and proband was 95.87% and 0 of the purified FXIIIA protein activity from the normal subject, respectively.</p><p><b>CONCLUSION</b>DelCD11-279 of FXIIIA mRNA which encoding a 464 amino acids of inactive FXIIIA protein is one of the molecular mechanisms resulting in FXIII deficiency in the patient.</p>

Identification of genetic defects in a Chinese pedigree with factor XIII deficiency: case report and literature review / 中华血液学杂志

<p><b>OBJECTIVE</b>To perform phenotypic diagnosis, genetic diagnosis and prenatal diagnosis of inherited coagulation factor XIII (FXIII)deficiency in a Chinese family also provide a review of inherited coagulation F XIII deficiency.</p><p><b>METHODS</b>The activity levels of F XIII (F XIII:C) of proband and family members were measured by clot solubility test and REA-chrom F XIII kit. The antigen levels of F XIII(FXIII:Ag) were measured by enzyme-linked immunosorbent assay. Thrombelastography (TEG) test was used to make a comprehensive evaluation of coagulation status in the proband. All 15 exons and exon-intron boundaries of the F13A1 gene were amplified by PCR, and DNA sequencing was performed then. The mutation identified in the proband was screened in the family members. Furthermore, the related literatures were reviewed to provide a profile of clinical manifestation, gene mutations, the relationship between the mutations and phenotype, and treatments of inherited coagulation F XIII deficient cases.</p><p><b>RESULTS</b>The clot solubility test was positive in the proband. The FXIII:Ag level of the proband was less than 1% and the FXIII:C level was below the lower limit of detection (<3%). Two compound heterozygous missense mutations (p.Arg662* and p.Trp665*) were identified in the proband. Family study showed that the two mutations were both inherited from the parents. The fetus also carried two compound heterozygous mutations, the same as the proband, and was diagnosed with severe F XIII deficiency. As reported in the literatures, most mutations were missense mutations and nonsense mutations, and no hot spot was found. The clinical pattern of F XIII deficiency varied among patients, with potentially fatal consequences from severe bleeding complications.</p><p><b>CONCLUSION</b>Better understanding of F XIII biochemical properties and function and developing of FXIII laboratory assays and genetic detection could prevent missed diagnosis, and patients moght benefit from better care.</p>

Recent review on blood transfusion therapy

Blood transfusion is an essential part of medical care, but it has risks, including infectious and immunologic complications. Recent medical practice emphasizes the rationalization of transfusion according to guidelines at the national and local levels. Early transfusions used whole blood, but modern practice commonly uses only components of the blood, such as red blood cells, platelets, plasma, and clotting factors. Red blood cell transfusions are indicated to improve oxygen delivery to tissues and to treat hemorrhage. Platelet transfusion may be indicated to prevent hemorrhage in patients with thrombocytopenia or functionally abnormal platelets. Fresh frozen plasma can be used to correct coagulation abnormalities in order to normalize the fibrinogen level, prothrombin time, and activated partial thromboplastin time. Cryoprecipitate is indicated for bleeding associated with fibrinogen deficiencies, factor XIII deficiency, hemophilia A, or von Willebrand's disease. However, blood transfusion should be based on guidelines as well as the patient's clinical condition. Appropriate use of blood components results in effective transfusion therapy and reduces transfusion-related complications.

Identification of a novel large deletion of factor subunit A mRNA associated with hereditary factor deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the expressed mRNA of the factor subunit A (FA) in monocyte in a hereditary factor (F) deficiency family.</p><p><b>METHODS</b>The F A mRNA of the proband and the other family members was analyzed by RT-PCR, semi-quantitative RT-PCR, cloning and sequencing. The three dimensional structure of the protein was predicted by SWISS-MODEL and viewed by RASMIOL.</p><p><b>RESULTS</b>(1) A large in frame deletion from codons 11 to 279, spanning from exon 2 to 7 of F A (DelCD11-279), was identified in the proband at mRNA level and a truncated protein is predicted composed of 464 amino acids. Compared with the normal and the other families, the proband showed lower level of F A mRNA in RT-PCR. (2) SWISS-MODEL analysis showed that the truncated protein lacked the β-sandwich and a part of catalytic core, resulting in loss of the normal catalytic domains.</p><p><b>CONCLUSION</b>DelCD11-279 of F A mRNA is associated with hereditary F deficiency. The reduced expressing level of F A gene is one of the causes resulting in F deficiency in the patients.</p>

Molecular study of coagulation factor XIII-A among patients with inherited factor XIII-A deficiency

Factor X[1] is the last enzyme in the clotting cascade. The gene of A chain is located on chromosome 6. Deficiency of factor XIII in autosomal recessive conditions occurs at a frequency of 1 in 2 million general population. The aim of this study was to detect the mutations of subunit A in both patients and carriers. In this study we have investigated the molecular basis of inherited FXIII deficiency among patients from 21 unrelated Iranian families. Mutation were detected by amplifying each exon. Those exons exhibiting the presence of hetero duplex formation sensitive gel electrophoresis, were selected for direct sequencing. After sequencing, detected mutation was carried out by restriction fragment length polymorphism [RFLP]. All patients having entered the study had mutations. Twelve patients had homologues substitution of TGG->CGG in exon 4, 1 insertion mutation occurring in exon 7 triple G, 2 patients demonstrated mutation exon 9 ATG-> AAG, 3 patients had substitution of CGG-> CAG in exon 10, and 3 patients showed a homologue subsituation mutation in exon 15 GCC->GTC. Our findings suggest that the activity of enzyme is highly dependent on the core domain. Changes in charge, amino acid tail and conformation lead to decreased enzyme activity. Also tetrameric structure is calcium related. It seems that changes of amino acid sequence convert enzyme stability

[Factor XIIIA-V34L and factor XIIIB-H95R gene polymorphisms in Shahrekord, 2010, I.R.Iran]

Some of the inherited coagulation factor polymorphisms have been related to the pathogenesis of venous thromboembolism and other adverse outcomes. As there are limited data on the prevalence of these polymorphisms in Iranian populations this study aimed to assess two factor XIII polymorphisms, FXIIIA-V34L and FXIIIB-H95R, in healthy individuals. In this cross sectional study 150 healthy blood donors from Shahrekord, Iran with no history of venous thromboembolism were recruited to the study. Genotyping from EDTA taken venous blood for the above polymorphisms was under taken by PCR - RFLP. Fifty one [34%] of participants were heterozygous for VL and 7[4.67%] were homozygous LL. 26 [17.33%] and 1[0.67%] were heterozygous and homozygous for RH and RR of FXIIIB respectively. 48.67% of the study population carried at least one of the above polymorphisms and there was no carrier of both as homozygous. The prevalence of these FXIII polymorphisms in healthy subjects is somehow similar to previously published data in Caucasian populations, but quite different than limited existing data from China and other ethnic groups. Such findings could be relevant to the ethnic similarities and differences

Molecular mechanisms of Arg77Cys missense mutation and Arg174stop nonsense mutation of factor XIII A gene causing severe factor XIII deficiency / 中国实验血液学杂志

The present study was aimed to investigate the molecular mechanisms responsible for the pathogenesis of severe factor XIII (FXIII) deficiency. Site-directed mutagenesis was conducted to obtain human FXIIIA expression plasmids bearing the mutations. Wild type FXIIIA recombinant plasmid (pcDNA3.1-FXIIIA-wt) and 2 mutant FXIIIA recombinant plasmids (pcDNA3.1/FXIIIA/77mut, pcDNA3.1/FXIIIA/174mut) were transfected into the cultured COS-7 cells using lipofectamine 2000 transfection reagent, respectively. FXIII activities were measured by the Berichrom(®) FXIII chromogenic assay. The expression levels of FXIIIA mRNA were detected by real-time RT-PCR. The recombinant FXIIIA mutants were determined by using Western blot and ELISA. The results showed that the normalized mRNA levels of 2 mutants in transfected COS-7 cells were 0.82 ± 0.21 and 0.76 ± 0.17, respectively. The relative levels of both mRNA transcripts were not significantly decreased as compared with the wild type (1.06 ± 0.51). FXIII activity and FXIIIA antigen levels in concentrated media of cell expressing the wild type protein were (24.0 ± 2.9)% and (13.2 ± 2.3)%, respectively. FXIII activity and FXIIIA antigen levels in cell lysates containing the wild type recombinant protein were (61.6 ± 30.4)% and (32.8 ± 14.5)%, respectively. However, the antigen levels and activity of 2 mutants were severely decreased as compared to the wild type. It is concluded that both mutations severely disturb the normal expression of FXIIIA protein. The reduction of expression levels and decreased activities of the 2 mutants provides a convincible explanation for the deficiency phenotype in the index case.

Frequency of bleeding disorders diagnosed at armed forces institute of pathology Rawalpindi

To determine the frequency of bleeding disorders diagnosed at Armed Forces Institute of Pathology, Rawalpindi [AFIP Rwp]. Descriptive study. Department of Hematology, AFIP Rwp from January 2006 to June 2009. A total of 1836 patients of bleeding diathesis were included in the study. Hess test was done to investigate the vascular defects. Bleeding Time [BT] was done to screen platelet function defects. The 'clotting screen' and mixing studies were done to detect coagulation protein defects. Clot solubility test was performed to screen factor XIII deficiency. Out of 1836 patietns of bleeding diathesis 435 [23.7%] were diagnosed as having haemostatic defects. Out of these 435 patients 273 [62.8%] had coagulation factor deficiency, 81 [18.6%] had platelet function defects and 81 [18.6%] had vWF deficiency. Among the 273 coagulation factor deficiency patients, factor VIII deficiency was in 121 [44.3%], factor IX deficiency in 32 [11.7%], factor V deficiency in 18 [6.6%], factor XIII deficiency in 15 [5.5%], factor VII deficiency in 12 [4.4%], factor X deficiency in 9 [3.3%], factor I deficiency in 8 [2.9%] and factor II deficiency was in 3 [1.1%]. Multiple factor deficiency was 55 [20.1%]. No defects of vasculature were identified. Coagulation factor deficiencies, with factor VII deficiency being the commonest are the most frequent bleeding disorders. Platelet function defects and vWF deficiency also comprise significant proportion of the bleeding disorders

Identification of Arg77Cys and Arg174stop double heterozygous mutation in a Chinese family with inherited FXIII deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the gene mutation type of an inherited coagulation factor XIII (FXIII) deficiency pedigree.</p><p><b>METHODS</b>PCR and DNA sequencing were used to identify the mutations in the 15 exons and the flank sequence of FXIII gene in the proband. The identified mutations were validated by allele specific PCR, PCR restriction fragment length polymorphism technique or DNA sequencing in the family members and 100 healthy volunteers.</p><p><b>RESULTS</b>Arg77Cys and Argl74stop double heterozygous mutations were discovered in the proband. The pedigree analysis showed that Arg77Cys missense mutation inherited from her father, and Arg174stop from her mother. The Arg77Cys missense mutation in exon 3 was not found in her husband and the other 100 healthy volunteers.</p><p><b>CONCLUSION</b>A novel Arg174stop nonsense mutation was discovered in human FXIII gene. A simple DNA assay based on PCR for detection of this mutation was developed. The congenital FXIII deficiency in the proband might be caused by the coinheritance of the Arg77Cys missense mutation in exon 3 and the Arg174stop nonsense mutation in exon 4.</p>

Hemorragia cerebral por déficit de factor XIII: a propósito de un caso / Cerebral hemorrhage factor XIII deficiency: a purpose of a case

La estabilidad del coágulo de fibrina es importante desde el punto de vista hemostático. La deficiencia del factor XIII (estabilizador de la fibrina) es un transtorno autosómico recesivo raro con una prevalencia calculada de uno en cinco millones. Se presenta caso de paciente masculino de 26 años de edad quien es portador de este transtorno, quien consulta por cefalea de fuerte intensidad en región hemicraneal derecha acompañado de episodios eméticos, sensación de vértigo sin rigidez de nuca. Es ingresado y realizan estudios imagenológicos RNM (Resonancia Magnética Cerebral con Fase de Angioresonancia) donde se visualiza foco hemorrágico intraparéquimatoso de gran tamaño a nivel de territorio de la cerebral media y otro de menor tamaño a nivel occipital derecha sin la evidencia de malformaciones arteriovenosas. El paciente no tiene buena evolución apareciendo nuevos focos hemorrágicos, el cual fué manejado por equipo multidisciplinario sin requerir acto quirúrgico.

Factor XIII deficiency in children-clinical presentation and outcome

To determine the demographic features and clinical outcome of children with Factor XIII deficiency. Observational case series. The Aga Khan University Hospital, Karachi, from January 1996 to December 2006. Records of all hospitalized pediatric patients with discharge diagnosis of FXIII D, on the basis of factor XIII assay 5 mol/L urea test were retrospectively reviewed and abstracted on a pre-specified proforma. Demographic features, coagulation profile, family history and outcomes were noted. A total of 10 charts were reviewed. There were 5 boys and 5 girls. Almost all the children [9/10] were less than 5 years of age, out of whom 5 [50%] were infants, and 3 were neonates. Bruises and prolonged bleeding after trauma was the major presenting complaints in 80%, followed by prolonged bleeding from the umbilical stump in 2 patients. Nine patients had past history of prolonged umbilical bleeding. Two patients had history of FXIII D in siblings, while 2 had history of prolonged bleeding in other family members [cause unknown]. Consanguinity was present in 80% of the families. Initial coagulation screen were normal in all patients. Two patients had intracranial hemorrhage, proved on neuro-imaging, were managed with plasma infusions and required craniotomy. The rest were managed conservatively with plasma transfusions. All were discharged alive in good clinical condition. Almost all were followed regularly in clinic with monthly cryoprecipitate transfusions. Although factor XIII deficiency is a rare genetic disorder in children with history of brui sing, prolonged umbilical bleeding, family history of bleeding and consanguinity with normal initial coagulation screen [PT, APTT and platelets], FXIII D should be ruled out

Identification of a novel mutation of F (13) A gene in a pedigree with factor XIII deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore F (13) A gene mutation in a pedigree with hereditary coagulation factor XIII (FXIII) deficiency.</p><p><b>METHODS</b>The FXIII deficiency was diagnosed by clot solubility test and other standard laboratory clotting tests. All exons, exon-intron boundary sequences of F(13) A gene were amplified by PCR and the products were sequenced directly. Any mutation identified by direct sequencing was confirmed by reverse sequencing. The mutation identified in the proband was screened in the family members.</p><p><b>RESULTS</b>The assays of PT, Qiulan, fibrinogen leveling, platelet counts, bleeding time were normal and the clot solubility test was positive in the proband. The homozygous deletion of 33 nucleotides (127067de133) in exon 10 of F(13) A gene which resulted in deletion of 11 amino acids in FXIIII A protein with 720aa residues was identified in the proband. Family studies showed that the mutation was inherited from the parents both of whom carried the heterozygous deletion mutation.</p><p><b>CONCLUSION</b>The homozygous 127067de133 mutation of F(13) A gene is responsible for the disorder of the pedigree.</p>

Factor XIII deficiency in a Kuwaiti child typical presentation with delayed diagnosis

Congenital factor XIII deficiency is a rare bleeding lack of a high index of suspicion. The patient was treated disorder, presenting usually during the neonatal period, successfully with cryoprecipitate and is doing well on We report here a case of a child with bleeding tendency cryoprecipitate prophylaxis. This is indeed, the first case due to congenital factor XIII deficiency. Though he had a report of this disorder from Kuwait typical presentation, the diagnosis was delayed due to lack of high index of surpecions. the patients was treated successfully with cryopricipitate and is doing well on cryopricipitate prophylaxis this is indeed, the first case report of this disorder from Kuwait

A novel genetic defect in a Chinese family with inherited coagulation factor XIII deficiency / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the genetic defect of inherited coagulation factor (F) deficiency in a Chinese family and to explore its molecular mechanism.</p><p><b>METHODS</b>The activity and antigen of plasma F were measured by photometric test and enzyme-linked immunosorbent assay, and rocket-electrophoresis, respectively. All the exons and exon-intron boundaries of the FA subunit gene were amplified by PCR and then DNA sequencing was performed. Restriction endonuclease analysis was used for the PCR products of the family members and 80 healthy donors to exclude gene polymorphism.</p><p><b>RESULTS</b>Rapid dissolution of the proband's fibrin clot occurred within 30 minutes, and antigen of his plasma F was significantly decreased, two compound heterozygous missense mutations (a C to T transition at nucleotide 177,246 which caused Arg703Trp, and a A to G transition at nucleotide 177,286 which caused His716Arg) in exon 15 of FA subunit gene were found. The possibility of gene polymorphism was excluded by restriction endonuclease analysing. Each of these two missense mutations was respectively found in his mother and father. Molecular modeling based on 3D crystallographic data predicted that the mutant protein decreased stability and was likely to be rapidly degraded.</p><p><b>CONCLUSIONS</b>The inherited F deficiency in the Chinese family is caused by two compound heterozygous missense mutations-Arg703Trp and His716Arg in the FA subunit, which to our knowledge, are reported for the first time.</p>

Combined occurrence of von Willebrand's disease and factor XIII deficiency: a case report.

We report the case of a three year old female child with combined occurrence of von Willebrand's disease and factor XIII deficiency, an extremely rare combination. The patient presented with prolonged bleeding following cuts and wounds. Clot solubility test using 5M urea was positive. Platelet aggregation using ristocetin was reduced, which corrected on adding normal plasma. Aggregation with other agonists was normal. We discuss the clinico- hematological profile of the case. Only one such case has been reported in literature in the past to the best of our knowledge.

A Case of Congenital Factor XIII Deficiency Diagnosed by Semiquantitative Method / 대한혈액학회지

Factor XIII (fibrin stabilizing factor) has roles of stabilizing clot and cross-linking fibrin polymer, so the congenital factor XIII deficient patient has clot unstability and delayed bleeding episodes. We experienced a case of 11 years old girl who had experienced delayed umbilical healing, several episodes of intraabdominal and intracranial hemorrhage. But her coagulation screening studies with prothrombin time, aPTT (activated partial thromboplastin time), bleeding time showed normal value at each episode. These findings suggested typical features of congenital factor XIII deficiency. We used semiquantitative method to diagnose Factor XIII deficiency and quantify Factor XIII. We has treated her successfully with prophylactic fresh frozen plasma through plasmapheresis from two donors.

Identification of two novel mutation in two Chinese hereditary coagulation factor XIII deficiency families / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore gene defect of hereditary coagulation factor XIII deficiency.</p><p><b>METHODS</b>PCR and gene sequencing or ARMS-PCR were used to detect the FXIIIA gene of peripheral white blood cell (PBC) from two Chinese hereditary coagulation factor XIII deficiency family members and 60 normal subjects respectively. The level of FXIIIA gene mRNA was tested by RT-PCR.</p><p><b>RESULTS</b>(1) Nucleotide sequence analysis of the two probands' and their family members' DNA revealed that all of the three patients had homozygous missense mutation in FXIII A subunit gene. Proband 1 had a C to G transition at nucleotide (nt) 1 241 in exon 10 and proband 2 and his sister a C to T transition at nt 232 in exon 3 of FXIII A gene, which resulted in the substitution of Ser413 with Trp and Arg 77 with Cys, respectively. Family study showed that the two mutations were inherited from the parents who were correspondingly heterozygotes at nt 1 241 or nt 232. (2) The two mutations were not found in the normal subjects. (3) The FXIIIA gene mRNA level in the two probands was a little decreasing.</p><p><b>CONCLUSION</b>It is the two novel mutations that results in FXIIIA deficiency. The two mutations of FXIIIA gene may affect its function or alter protein folding. The defective FXIII which is unstable and degraded rapidly in cytoplasm may be the main cause of FXIII deficiency.</p>