Assessing Gene Expression of factor XIII-A in Iraqi patients with FXIII deficiency

Coagulation factor XIII (FXIII) circulates in plasma as a pro-transglutaminase heterotetrameric complex (FXIIIA2B2), which upon activation by thrombin and calcium covalently crosslinks preformed fibrin polymers. The heterotetrameric complex is composed of a catalytic FXIIIA2 subunit and a protective/regulatory FXIII-B2 subunit coded by F13A1 and F13B genes, respectively. The study aimed to assess the level of expression of the FXIII-A gene in seven Iraqi families affected with FXIII deficiency. Samples of whole blood were collected from seven Iraqi families that were characterized with F13 deficiency register in the Hemophilia Ward, Children Welfare Teaching Hospital, Medical City, Baghdad. These samples were divided into three groups according to the genotype, which included affected homozygous recessive and heterozygous with control healthy group. RNA was isolated from whole blood samples and gene expression levels of the F13 A1 gene was determined using quantitative Real-Time polymerase chain reaction (RT-qPCR), the result revealed that the expression levels of the F13A gene in heterozygous parents were significantly increased compared with the homozygous affected and control group. The (ROC) curve was analyzed in this study to determine the optimal cut-off value for F13 expression and showed a very good predictor for FXIII deficiency diagnosis with "Area under the ROC Curve." AUC equals 0.844 and p< 0.001.


Introduction
Coagulation factor XIII (FXIII) is a protein from the group of transglutaminases (TGase) that has two catalytic A subunits (FXIII-A) and two transporter noncatalytic B subunits (FXIII-B) in form (A2B2) (1).It is a proenzyme that is activated by thrombin that is generated in the final stage of the blood coagulation cascade (2).The A subunit has the main function, and it includes the catalytic core domain, activation peptide, calcium ion binding site, and other structure domains.The B subunit mainly acts as a carrier protein to stabilize the A subunit, connects the A subunit to fibrinogen, and downregulates the activity of FXIII (3).FXIII-A is synthesized primarily in cells of bone marrow origin, while FXIII-B is synthesized in the liver.The formation of tetrameric complex probably occurs in plasma (4).Coagulation Factor XIII (FXIII) plays an important role in wound healing by stabilizing fibrin clots and cross-linking extracellular matrix proteins (5).Mature FXIII-A is a 731-amino acid protein without a hydrophobic leader sequence (6).FXIII deficiency is one of the blood bleeding disorders that results from the deficiency or functional abnormality of one of the plasma proteins involved in providing normal coagulation that poses a high risk for severe hemorrhage (7).Bleeding usually includes umbilical bleeding, prolonged bleeding after an injury or surgical procedure, subcutaneous bleeding, gum bleeding, intracranial bleeding, joint bleeding, and muscle bleeding (8).(10).The gene coding for the A subunit has been localized to chromosome 6p24-25.
Hereditary deficiency of coagulation factor XIII is inherited as an autosomal recessive trait (1).This study Assesses the mRNA level of coagulation factor XIII-A in whole blood using a one-step realtime-quantitative polymerase chain reaction (q-Rt PCR) and compares the assessing Gene Expression among three groups that were identified.All the laboratory tests were performed at the Hemophilia Ward, Children Welfare Teaching Hospital, Medical City, Baghdad laboratory.Five ml of blood was collected from seven families via venipuncture and transferred to a tube containing triazole in volume (1:2), the general characterization features of the seven families are shown in Table 1.

Real-Time Polymerase Reaction (RT-qPCR)
Total RNA was extracted from the blood of available

General characteristic of the study
The study included a total of seven families, containing parents and children, in addition to thirty healthy individuals.Table 3 shows all the details of the families.
The families under study were classified according to the clinical condition into affected individuals who were characterized by bleeding symptoms as a result of factor 13 deficiency, while the other category included healthy parents without symptoms with a bleeding disorder called heterozygosity because the disorder is a pattern of recessive inheritance.

Expression of F13A gene
The current study analyzed the mRNA expression levels of the FXIII A1 gene in whole blood samples of all families with FXIII deficiency patients and controls by Rt q PCR.Relative FXIII A1 expression levels of patients were obtained by using β-Globin as a reference for the gene for normalization and HCs as a calibrator sample.Cycle threshold Ct values were calculated by the 2-∆∆Ct method for evaluation of the expression levels.
The results indicated that the expression levels of the F13A gene found in patients were high expression.
The median expression fold of the control group was 1.00, but it was observed that the median expression fold of affected and heterozygous individuals increased (1.3, 3.6) with a property value equal to 0.001.It is important to note that the median expression fold of heterozygote individuals was higher than that of those affected with FXIII deficiency compared to the control group, as shown in Table 4.

Diagnostic value of the expression of FXIII gene
To understand whether the expression level of the FXIII gene in blood could differentiate patients from healthy controls, a Receiver Operating Characteristic (ROC) curve analysis was conducted.The result showed that the gene expression levels of F13A in patients had a very good ability to differentiate patients from healthy individuals.The AUC, sensitivity, and specificity were 0.844, 84.37%, and 100.00 %, respectively, at the cutoff value of >1 fold, which was the good value of FXIII deficiency correct prediction, as shown in Figure 1.Our results of ROC curve analysis showed that gene expression levels of F13A in patients could represent a very good predictor for FXIII deficiency diagnosis.
Whole blood was collected from seven Iraqi families, whose members that diagnosed and registered with congenital FXIII deficiency in the Hemophilia Ward, Children Welfare Teaching Hospital, Medical City, Baghdad.The demographic and clinical features and treatment have been collected for included patients, and they were diagnosed by having bleeding tendency and normal standard coagulation tests (normal platelet count, normal prothrombin time, no normal partial thromboplastin time, and normal bleeding time), and the diagnosis was confirmed by clot solubility test in 5 m urea (qualitative test for FXIII deficiency).
families' individuals and healthy controls using the protocol of TRIzol™ Reagent, Quantus Fluorometer was used to detect the concentration of extracted RNA to detect the quality of samples for downstream applications.Analysis and Calculation of gene expression levels of one or more genes depend on RNA concentration after conversion to cDNA.By using GoTaq® 1-Step RT-qPCR System, MgCL2, Nuclease Free Water; Promega, USA.primers for a target gene and housekeeping gene were supplied by Macrogen Company in a lyophilized form.and the desired sequence was blasted in the relevant database (http://www.ncbi.nlm.nih.gov/pubmed), the sequence and details of primers are shown in Table -2.The results of RT-qPCR were analyzed by the relative quantification of gene expression level (fold change) according to Livak (12).Based on comparing the distinct cycle determined by threshold values Ct at a constant fluorescence level.The target gene was normalized to an endogenous control (HKG) and relative to the calibrator which is the target gene in the healthy control group.The fold-change was calculated for each sample using the following equations: ΔCt sample = Ct gene -Ct HKG, ΔΔCt sample= (ΔCt sample)-(average ΔCt control group), Fold-change sample= 2-ΔΔCt.

Primer Name Sequence 5 `
for the Social Sciences (SPSS) version 22 has been used in the current study for dealing with obtained data.Mean (median) and interquartile range have been used to calculate parametric results(quantitative) while nonparametric data(qualitative) was calculated by means and standard deviation.Additionally, the Pearson Chi-square test was used for comparisons, and the Spearman correlation was used to test the correlation between different study parameters.The P-value is significant < 0.05 at a confidence interval of 95%.

Figure 1 :
Figure 1: Receiver operator curve (ROC) analysis for the predictive value of relative expression of FXIII A1 levels (fold) in patients versus healthy controls.AUC= area under the curve, SE= standard error, CI= confidence interval, PPV = positive predictive value, NPV= negative predictive value.

Table 3 :
General Characteristics of patients with FXIII deficiency and control N= Frequency, %= Percentage

Table 4 :
Relative expression of F13A levels (fold) in blood samples of the studied groups.