Analysis of four hereditary protein C deficiencies associated with vascular thromboembolism.

OBJECTIVE: To analyze the clinical features and gene mutations in four families with hereditary protein C (PC) deficiency and explore their association with vascular thromboembolism. METHODS: The clinical data of four patients with PC deficiency were retrospectively analyzed. Venous blood samples were collected from the four affected patients and their family members, and relevant coagulation indexes and thrombin production and inhibition tests were performed. PCR was used to amplify and directly sequence the PROC gene of the probands. Software analysis was conducted to assess the conservativeness and pathogenicity of the mutated loci. Protein models were constructed to analyze the spatial structure before and after the mutation. RESULTS: Thrombin generation and inhibition assays demonstrated impaired anticoagulation in all four probands. Proband 1 and 4 presented clinically with pulmonary embolism and lower extremity deep vein thrombosis (DVT), Proband 2 with cerebral infarction, and Proband 3 with DVT. Genetic analysis revealed the presence of the following mutations: c.541T > G heterozygous missense mutation, c.577-579delAAG heterozygous deletion mutation, c.247-248insCT heterozygous insertion mutation, c.659G > A heterozygous missense mutation, and a new variant locus c.1146_1146delT heterozygous deletion mutation in the four probands, respectively. In particular, c.1146_1146delT heterozygous deletion mutations not reported previously. Conservativeness and pathogenicity analyses confirmed that most of these amino acid residues were conserved, and all the mutations were found to be pathogenic. Analysis of protein modeling revealed that these mutations induced structural alterations in the protein or led to the formation of truncated proteins. According to the American College of Medical Genetics and Genomics (ACMG) classification criteria and guidelines for genetic variants, c.1146_1146delT was rated as pathogenic (PVS1 + M2 + PM4 + PP1 + PP3 + PP4). CONCLUSION: The identified mutations are likely associated with decreased PC levels in each of the four families. The clinical manifestations of hereditary PC deficiency exhibit considerable diversity.

New plasma protein C and protein S concentrate: A synergy for therapeutic purposes.

BACKGROUND AND OBJECTIVES: Deficiencies of protein C (PC) or protein S (PS) are rare diseases, characterized by mutations in the PC or PS genes, which encode plasma serine proteases with anti-coagulant activity. Severe PC or PS deficiencies manifest in early life as neonatal purpura fulminans, a life-threatening heamorrhagic condition requiring immediate treatment. First-line treatment involves replacement therapy, followed by maintenance with anti-coagulants. Replacement therapy with specific protein concentrates is currently only limited to PC, and therefore, a PC + PS concentrate represents a useful addition to therapeutic options, particularly for severe PS deficiency. Further, the production of a PC + PS concentrate from unused plasma fractionation intermediates would impact favourably on manufacturing costs, and consequently therapy prices for patients and health systems. MATERIALS AND METHODS: Several chromatographic runs were performed on the same unused plasma fractionation intermediates using different supports to obtain a PC/PS concentrate. The best chromatographic mediums were chosen, in terms of specific activity and recovery. A full process of purification including virus inactivation/removal and lyophilization steps was set up. RESULTS: The final freeze-dried product had a mean PC concentration of 47.75 IU/mL with 11% of PS, and a mean specific activity of 202.5 IU/mg protein, corresponding to over 12,000-fold purification from plasma. CONCLUSION: The development of a novel concentrated PC/PS mixture obtained from a waste fraction of other commercial products could be used for its potential therapeutic role in the management of neonatal purpura fulminans pathology.

Thrombosis-related characteristics of pregnant women with antithrombin deficiency, protein C deficiency and protein S deficiency in Japan.

BACKGROUND:  We previously conducted a primary survey of pregnant women with hereditary thrombophilia based on national surveillance in Japan, but did not examine their thrombosis-related characteristics. Antithrombin (AT) deficiency, protein C (PC) deficiency and protein S (PS) deficiency are the major types of hereditary thrombophilia in Japan. METHODS: We examined their detailed information related to thrombosis, and evaluated peripartum outcomes in comparison with control data obtained from the Japan Society of Obstetrics and Gynecology. RESULTS: Definite or probable AT deficiency, PC deficiency and PS deficiency were observed in 80, 50, and 317 pregnancies, respectively, from 2014 to 2018 in Japan, with prevalence rates among total deliveries of 0.011%, 0.007%, 0.044%. The number of pregnancies with AT, PC and PS deficiency might have been as many as 27, 17 and 108 every year if complete answers had been provided. In the peripartum period of current pregnancies, 27.5% of women with AT deficiency, 28.0% with PC deficiency and 13.2% with PS deficiency developed thrombosis (p < 0.001 vs. control). Pregnant women with AT and PC deficiency were more susceptible to thrombosis than those with PS deficiency (P < 0.01). Of the thromboses, 92.3% occurred during pregnancy, 62.8% at less than 15 gestational weeks. The earliest onset of thrombosis was 5 gestational weeks. Prophylactic anticoagulation significantly prevented the onset of both antepartum and postpartum thrombosis (p < 0.0001). The rate of recurrent pregnancy loss in women with low PC or PS activities was significantly higher than in controls (p < 0.0001); however, it is unknown whether recurrent pregnancy loss is related to hereditary PS deficiency. There seem to have been few serious maternal or fetal/neonatal complications due to placental insufficiency related to a hypercoagulable state other than growth restriction. CONCLUSIONS: This survey revealed the thrombosis-related characteristics of pregnant women with hereditary thrombophilia in Japan. We suggest prophylactic anticoagulation to prevent maternal or fetal/neonatal complications.

Two cases of venous thromboembolism in siblings after splenectomy due to a novel PROC gene mutation.

BACKGROUND: Venous thromboembolism(VTE)is a common multifactorial disease. Anticoagulant protein deficiency is the most usual hereditary thrombophilia in the Chinese people, which includes protein C(PC), protein S and antithrombin deficiencies. CASE PRESENTATION: A retrospective analysis was conducted on clinical manifestations, laboratory tests, genetic information, and other relevant data of siblings diagnosed with VTE in 2020 at the Department of Pediatrics of Shenzhen Second People's Hospital. The proband, a 12-year-old female, was admitted to the hospital in December 2020 with a complaint of pain in the left lower limb for four days. The examination found that the PC activity was 53%, and B-ultrasound showed bilateral thrombosis of the great saphenous vein in the thigh segment. The proband's younger brother, a 10-year-old male, was admitted to the hospital in January 2021 due to right lower limb pain for two weeks. PC activity is 40%. B-ultrasound showed superficial venous thrombosis in the left lower limb and upper limb. Both siblings suffered from thalassemia and underwent splenectomy before recurrent thrombosis occurred. The proband's mother was asymptomatic, and her PC activity was 45%. Both cases were treated with warfarin anticoagulation, and their symptoms improved. The proband's mother was found to have a heterozygous mutation at this locus through Sanger sequencing. CONCLUSION: Protein C deficiency should be considered for venous thromboembolism in childhood. The heterozygous mutation 1204 A > G in PROC exon 9 in this family is reported for the first time.

[Perioperative management for fracture in a child with homozygous congenital protein C deficiency].

Congenital protein C (PC) deficiency is one type of hereditary thrombosis. Patients with hereditary thrombosis are at high risk for thrombosis in the perioperative period, but a standard management strategy has not been established. Here we report a case of perioperative management of a fracture in a child with homozygous congenital PC deficiency. The patient was a 3-year-old boy who was diagnosed with congenital PC deficiency at birth. He sustained a traumatic supracondylar fracture of the right humerus and underwent emergency surgery. To prepare for open surgery for fixation of the fracture, warfarin was discontinued, and an activated PC (APC) concentrate was used in combination with vitamin K antagonism. However, warfarin was administered during the scheduled nail extraction because the operation was minimally invasive. No thrombotic or bleeding complications occurred in either operation. In emergency surgery in patients with congenital PC deficiency, the combination of vitamin K and APC concentrate is considered a maintenance option for PC deficiency. Postoperative PT-INR control was difficult in our patient due to the administration of vitamin K and withdrawal of warfarin, and this issue must be addressed in the future. Further case experience is desirable to standardize perioperative management.

[Extracorporeal Membrane Oxygenation Combined With Interventional Thrombectomy for Treating High-Risk Pulmonary Embolism Caused by Protein C Deficiency:Report of One Case].

Hereditary protein C deficiency is a chromosomal genetic disease caused by mutations in the protein C gene,which can lead to venous thrombosis and is mostly related to mutations in exons 4-9 and intron 8.Fatal pulmonary embolism caused by mutations in the protein C gene is rare,and the treatment faces great challenges.This article reports a case of fatal pulmonary embolism caused by a frameshift mutation in exon 8 of the protein C gene and summarizes the treatment experience of combining extracorporeal membrane oxygenation (for respiratory and circulatory support) with interventional thrombectomy,providing a basis for the diagnosis and treatment of this disease.

Evaluation of prothrombotic risk of two PROC hotspot mutations (Arg189Trp and Lys193del) in Chinese population: a retrospective study.

BACKGROUND: R189W and K193del of protein C (PC) were hotspot mutations in Chinese population with venous thromboembolism (VTE), but almost two-thirds of patients with above mutations coexisting with other genetically or aquiredly prothrombotic risk factors. The aim of this study is to clarify the independent contributions of R189W or K193del to VTE risk. METHODS: 490 unrelated patients with a personal history of VTE and 410 healthy participants were enrolled in this study. Data of their demographics, family history, genetic and acquired thrombosis risk factors were collected and statistically analyzed. RESULTS: PC R189W and K193del were identified in 3/410 (0.7%) and 7/410 (1.7%) healthy controls, and in 27/490 (5.5%) and 43/490 (8.8%) patients with VTE, respectively. Notably, about 70% of these mutant carriers combined with other genetic or acquired thrombophilic factors. After adjustment for age, gender, other inherited and acquired risk factors, we demonstrated that R189W and K193del were associated with 5.781-fold and 4.365-fold increased risk of VTE, respectively, which were significantly lower than the prothrombotic risk of anticoagulant deficiencies induced from rare mutations. Independent R189W or K193del mutation was not associated with earlier first-onset age as well as higher recurrent rate of VTE. However, combination of other genetic or acquired thrombophilic factors had supra-additive effects on those consequences. The more additional risk factors the patients had, the younger first-onset ages and higher risk of recurrence would be. CONCLUSIONS: As the most frequent mutations for PC deficiency in Chinese population, both R189W and K193del mutations had limited independent contributions to VTE development compared with other rare mutations in PROC gene, but may act in concert with other genetic defects or acquired thrombotic risk factors to produce the final severe phenotype.

Successful perioperative management using prothrombin complex concentrates in patients with severe congenital protein C deficiency.

Perioperative management of severe congenital protein C deficiency remains unestablished. This deficiency is often treated with anticoagulants, such as warfarin. Although anticoagulants need to be perioperatively discontinued, there are few methods for the management of such patients. We adopted a method for administering prothrombin complex concentrates (PCC), which includes intermittent administration of inactive protein C (PPSB-HT), and examined its outcome as a perioperative management approach for severe congenital protein C deficiency. Three patients underwent our perioperative management six times. We monitored activity levels of protein C, factor IX, and so forth. These patients could be perioperatively managed with PCC treatment.

Ghrelin, growth hormone and insulin-like growth Factor-I levels in people with protein C deficiency.

The primary objective of this study was to examine if people with protein C deficiency, which is a natural anticoagulant and also an endogenous acyl ghrelin peptidase, have elevated circulating levels of acyl ghrelin. The clinical trial was conducted in a university hospital setting. People with protein C deficiency were identified and invited to participate by a specialized coagulation outpatient clinic. People with protein C deficiency were examined and compared to age, sex, and body mass index matched healthy controls with regards to acyl ghrelin, unacylated ghrelin, growth hormone (GH) and insulin-like growth-factor I (IGF-I) in a cross-sectional case-control study. Systemic levels of acyl ghrelin, desacyl ghrelin, acyl-to-desacyl ghrelin ratio, GH and IGF-I were similar in people with protein C deficiency and healthy controls. Despite a significant reduction of protein C in people with protein C deficiency, there was no difference in acyl ghrelin or the secondary end points unacylated ghrelin, GH, or IGF-I in people with protein C deficiency.

A case of polycystic ovary syndrome with inevitable miscarriage and multi-site venous thrombosis caused by hereditary protein C deficiency.

BACKGROUND: Polycystic ovarian syndrome (PCOS) affects up to 18% of reproductive-aged women and raises the risk of venous thromboembolic disease (VTE), due to metabolic features and an apparent fibrinolytic state. Recent studies have shown an increased risk of VTE (1.5- to 2-fold) in patients with PCOS as compared to those without PCOS. Mutations in the Protein C (PC) gene (PROC) lead to deficiency or dysfunction of the protein, Protein C deficiency is the main clotting physiological inhibitor of protein C cofactors, and is a risk factor for venous thrombosis, which can cause a variety of events, including miscarriage. This case report proposes a correlation between PCOS, protein C deficiency, venous thrombosis and inevitable miscarriage. CASE PRESENTATION: A 33-year-old Chinese woman was diagnosed with Polycystic Ovary Syndrome (PCOS) in 2015. During the course of treatment, she took ethinylestradiol and cyproterone acetate tablets for more than one year. In 2016, she was sent to a hospital for emergency care due to explosive thrombosis (thrombosis in multiple parts of the body and pulmonary thrombosis). In 2020, the patient became pregnant via natural means and came to our hospital for treatment. During the second trimester, she experienced an inevitable miscarriage. High-throughput sequencing (NGS) of peripheral blood lymphocytes revealed that the patient had a protein C deficiency resulting from a heterozygous mutation deletion of 572_574 in exon 7. CONCLUSION: PC deficiency in conjunction with PCOS and the concomitant use of oral contraceptive (COC) would increase the risk of VTE, especially in the early stages of COC use.

[Clinical guidance for the peripartum management of patients with hereditary thrombophilia].

Hereditary thrombophilia is a condition in which individuals are susceptible to the formation of thrombi due to a hereditary deficiency in the anticoagulant factors antithrombin (AT), protein C, or protein S. The recommendations for peripartum management are as follows: 1) For women with acute venous thromboembolism (VTE) during pregnancy, anticoagulant therapy with a therapeutic dose of unfractionated heparin (UFH) is recommended; 2) For women with a history of VTE, a prophylactic dose of UFH is suggested during pregnancy; 3) For those with AT deficiency, supplementation with an AT preparation in addition to UFH is suggested; 4) For women with no history of VTE, anticoagulant therapy during pregnancy is considered for each thrombophilia type; 5) When anticoagulant therapy consisting of a prophylactic dose of UFH is administered during pregnancy, injection is discontinued with the onset of labor pains in cases of vaginal delivery and 6 hours before the start of delivery in cases of planned delivery or cesarean section; 6) In cases of AT deficiency, regardless of the thrombophilia type, supplementation with AT before and after delivery is suggested; 7) For women with thrombophilia and a history of VTE and for those who receive anticoagulant therapy during pregnancy, postpartum anticoagulant therapy is recommended.

Severe protein C deficiency in a newborn caused by a homozygous pathogenic variant in the PROC gene: a case report.

BACKGROUND: Severe protein C deficiency is a rare and inherited cause of thrombophilia in neonates. Protein C acts as an anticoagulant, and its deficiency results in vascular thrombosis. Herein, we report a case of protein C deficiency with a homozygous pathogenic variant in a term neonate, with good outcomes after proper treatment. CASE PRESENTATION: A four-day-old male newborn was transferred to the Seoul National University Hospital on account of dark red to black skin lesions. He was born full-term with an average birth weight without perinatal problems. There were no abnormal findings in the prenatal tests, including intrauterine sonography. The first skin lesion was observed on his right toes and rapidly progressed to proximal areas, such as the lower legs, left arm, and buttock. Under the impression of thromboembolism or vasculitis, we performed a coagulopathy workup, which revealed a high D-dimer level of 23.05 µg/ml. A skin biopsy showed fibrin clots in most capillaries, and his protein C activity level was below 10%, from which we diagnosed protein C deficiency. On postnatal day 6, he experienced an apnea event with desaturation and an abnormal right pupillary light reflex. Brain computed tomography showed multifocal patchy intracranial hemorrhage and intraventricular hemorrhage with an old ischemic lesion. Ophthalmic examination revealed bilateral retinal traction detachments with retinal folds. Protein C concentrate replacement therapy was added to previous treatments including steroids, prostaglandin E1, and anticoagulation. After replacement therapy, there were no new skin lesions, and the previous lesions recovered with scarring. Although there were no new brain hemorrhagic infarctions, there was ongoing ischemic tissue loss, which required further rehabilitation. Ophthalmic surgical interventions were performed to treat the bilateral retinal traction detachments with retinal folds. Molecular analysis revealed a homozygous pathogenic variant in the PROC gene. CONCLUSION: Severe protein C deficiency can manifest as a fatal coagulopathy in any organ. Early diagnosis and proper treatment, including protein C concentrate replacement, may improve outcomes without serious sequelae.

Clinical guidance for peripartum management of patients with hereditary thrombophilia.

Hereditary thrombophilia is a condition in which individuals are susceptible to the formation of thrombi due to a hereditary deficiency in anticoagulant factors, antithrombin (AT), protein C (PC), or protein S (PS). Many Japanese thrombophilia patients have PS deficiency, especially PS p.K196E (also called as PS Tokushima), which is exclusive to the Japanese population, and thrombosis sometimes occurs during pregnancy. At present, no management guidelines for pregnancy and delivery in thrombophilia patients have been developed. The Study Group for Hereditary Thrombophilia, one of the research groups of blood coagulation abnormalities in the Research Program on Rare and Intractable Diseases supported with the Research Grants of the Ministry of Health, Labour and Welfare Science, has therefore developed this clinical guidance to provide healthcare workers with necessary information on safe pregnancy, parturition and neonatal management, adopting a format of responses to seven clinical questions (CQ). At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Recurrent Cerebral Venous Thrombosis Treated with Direct Oral Anticoagulants in a Japanese Man with Hereditary Protein C Deficiency.

We herein report a case involving a 32-year-old Japanese man with recurrent cerebral venous thrombosis due to hereditary protein C deficiency. He was admitted to our hospital with impaired consciousness. Brain magnetic resonance imaging demonstrated high intensities diffusely along the bilateral sulci and magnetic resonance venography revealed left transverse sinus and superior sagittal sinus stenoses. His father had a history of cerebral infarction and venous thrombosis. The protein C activity level examined by chromogenic synthetic substrate assay was markedly reduced. He was diagnosed with protein C deficiency, and a genetic analysis revealed a heterozygous mutation at exon 3 c.199G>A,p.Glu67Lys on the protein C gene. Four months later, at his second admission, he had transient aphasia, and his protein C activity was under 10%. We switched warfarin to the direct oral anticoagulants edoxaban. He remains fully recovered with no adverse events after the administration of edoxaban for a year. Direct oral anticoagulants may be a new tool for treating cerebral venous thrombosis due to hereditary protein C deficiency.

[Thrombosis of the inferior vena cava and right atrium in a neonate with thrombophilia: diagnosis, treatment, result]. / Tromboz nizhnei poloi veny i pravogo predserdiia u novorozhdennogo s trombofiliei: diagnostika, lechenie, rezul'tat.

Hereditary thrombophilia is rare pathology giving rise to a ninefold increase in the risk for the development of thromboembolism in infants. The problem is multifactorial and characterized by high mortality, especially in neonates. Infants who develop thrombosis, particularly those with no family history, are often subjected to testing for hereditary thrombophilia. However, genetic testing for thrombophilia does not change the plan of treatment but makes it possible to perform prevention of thrombosis within the risk periods for the patient. Poor awareness of paediatricians, the complexity of carrying out genetic testing, the absence of approaches supported by evidence-based medicine due to shortage of high-quality clinical trials and no guidelines on prevention of thromboembolism in infants, as well as the frequent occurrence of diversified causes and diseases in different age groups make the problem significant for modern medicine. Further studies are needed to address many unanswered as yet questions.

Ophthalmic manifestations of congenital protein C deficiency: a case report and mini review.

BACKGROUND: Homozygous protein C (PC) deficiency is a potentially fatal disease with ocular blinding presentation or sequela. CASE PRESENTATION: A 5 month-old boy was presented for evaluation of leukocoria. He had a history of frequent bruises and PC deficiency, treated with warfarin. His intraocular pressure was normal. In the left eye leukoma with anterior segment dysgenesis, shallow anterior chamber, and cataract were observed. Fundus was not visible. B-scan revealed a closed funnel retinal detachment. His right eye had a normal anterior segment and a thin retina with anomalous retinal vascular branching at equator and peripheral retina. A fibrovascular tuft on the optic nerve head with induced traction on superior arcade was visible. Total loss of a and b wave of both were appreciated in electroretinography (ERG). Fluorescein angiography (FA) showed very severe leakage at the junction of the vascularized and non-vascularized retina and optic nerve head. Favorable outcome was achieved with lasering of avascular retina in the right eye. CONCLUSION: The potential for protein C deficiency should be assessed in all infants with leukocoria, anterior segment dysgenesis, retinal detachment and retinal dysplasia. Early diagnosis could save the child's life and vision.

Management of severe congenital protein C deficiency with a direct oral anticoagulant, edoxaban: A case report.

A male patient diagnosed with severe congenital protein C (PC) deficiency during the neonatal period was treated with long-term warfarin but frequently developed purpura fulminans and bleeding. At four years of age, edoxaban was initiated (direct oral anticoagulant [DOAC]). His d-dimer and fibrin/fibrinogen degradation product levels were closely monitored. His PC activity increased from below the sensitivity range to 17%; this increase was thought to be due to a reduction in PC consumption during edoxaban therapy. After edoxaban introduction, he experienced just one episode of purpura fulminans over two years without any adverse events. Thus, DOAC may be a promising alternative for the management of congenital PC deficiency.

Hereditary protein C deficiency caused by novel compound heterozygous mutants in a Chinese pedigree: A case report.

Purpura fulminans (PF) is a neonatal presentation of homozygous or compound heterozygous protein C (PC) deficiency; infants who are diagnosed with it are determined to have a major defect in coagulation regulation which is associated with undetectable levels of PC. We report a pedigree who suffered from the hereditary PC deficiency with compound heterozygous mutants; genetic analysis revealed compound heterozygous mutations of 262 G > T(Asp88Tyr) and 400 + 5G > A that were identified in the proband; moreover, Asp88Tyr and 400 + 5G > A were also detected in the father and the mother, respectively. A bioinformatics analysis revealed 262 G > T is probably damaging, and structural analysis indicated a possible mechanism for the functional impairment of PC in this pedigree.

Clinical factors associated with pregnancy outcome in women with recurrent pregnancy loss.

The aim of this prospective cohort study was to evaluate clinical factors associated with pregnancy outcomes in women with recurrent pregnancy loss (RPL). Women with a history of two or more pregnancy losses underwent workups for clinical factors of RPL and their pregnancies were followed-up with informed consent. Two hundred eleven (81.5%) of 259 women with RPL became pregnant. The multivariable analyses demonstrated that age (p < .01, OR 0.9, 95%CI 0.97-0.83), uterine abnormality (p < .05, OR 0.3, 95%CI 0.11-0.8), and protein C (PC) deficiency (p < .01, OR 0.14, 95%CI 0.03-0.6) were independent factors for becoming pregnancy in women with RPL. The number of previous pregnancy loss (p < .01, OR 0.57, 95%CI 0.43-0.75) and natural killer (NK) cell activity ≥33% (p < .01, OR 0.31, 95%CI 0.13-0.73) were independent factors for live birth in the subsequent pregnancy. Advanced age, the presence of uterine abnormality, and PC deficiency were risk factors for reduced pregnancy rate in women with RPL. Increased number of previous pregnancy loss and high NK cell activity were risk factors for miscarriage in the subsequent pregnancy. These results involve important information and are helpful for clinical practitioners.

Searching for genetic variants associated with thrombophilia.

Thrombotic states are inherited or acquired predisposition for thrombosis in the human vascular system. Nowadays Leiden mutation and mutation in prothrombin G20210A contributing to congenital thrombophilia are routinely tested. These mutations have a high prevalence in the population. Congenital deficiencies of protein S, protein C and antithrombin III are rare thrombophilia with lower population frequency, but higher risk of thromboembolic event. The genetic causes are mutations in the genes, which encode these proteins. The choice of proper molecular genetic testing depends on the difference in the detection of well-known single nucleotide polymorphism or unknown/rare variant. For the detection of causative variant FV Leiden and prothrombin G20210A are mostly used PCR-RFLP, reverse Strip Assay®, allele-specific PCR, TaqMan real-time PCR and SNaPshot®. Precise patient selection should precede the genetic testing of rare variants in anticoagulant proteins. It is appropriate to use methodology of massive parallel sequencing supplemented by a methodology for the detection of larger gene rearrangements - MLPA. We are successfully employing this approach in our institute. This methodology is faster with larger analytic capacity compared to commonly used direct sequencing by Sanger method.