Figure 4 - available via license: Creative Commons Attribution-NonCommercial 4.0 International
Content may be subject to copyright.
Identification of a novel missense mutation in coagulation factor IX c.855G>C, p. (E285D) causing hemophilia B in Saudi Arabian patient. (A) Family pedigree. (B) PCR gel image of factor IX exon 8, showing single specific band of product size 707 bp in two patients (lanes 1 and 2). (C) Representative electrophoregram showing WT genotype. (D) Representative electrophoregram showing the missense mutation (conflict) in exon 8 of factor IX.
Source publication
Background
Hemophilias A and B are X-linked bleeding disorders caused by mutations in the factor VIII and factor IX genes, respectively. Our objective was to identify the spectrum of mutations of the factor VIII and factor IX genes in Saudi Arabian population and determine the genotype and phenotype correlations by molecular dynamics (MD) simulatio...
Context in source publication
Context 1
... patient was a 1-month-old male with episodic bleeding and severe form of hemophilia B, he was unable to flex his knees, elbows and forearms, and he was undergoing treatment with factor IX concentrates. The electrophoregram is shown in Figure 4 Table 2, but figures for the electrophoregrams and PCR gel pictures of these known mutations were not shown here. ...
Similar publications
Compensated pathogenic deviations (CPDs) are sequence variants that are pathogenic in humans but neutral in other species. In recent years, our molecular understanding of CPDs has advanced substantially. For example, it is known that their impact on human proteins is generally milder than that of average pathogenic mutations and that their impact i...
Citations
... Hemophilia A (HA) and hemophilia B (HB) (OMIM: 306700 and 306900, respectively) are X-linked recessive bleeding disorders that are caused by the inheritance of genetic variants affecting F8 and F9 genes, those encoding coagulation factors VIII and IX with a consequent deficiency or dysfunction for its relevant factor. 1 The F8 gene sited at the end of the long arm of the X chromosome, at Xq28, is extremely large and structurally complex (186 932 bp; nearly 180-kb length containing 26 exons), while the F9 gene placed more toward the centromere, at Xq27.6, is considerably smaller and structurally simpler (nearly 34-kb length containing only 8 exons). 2 HA is more prevalent than HB worldwide. 3 In 2021, a meta-analysis study suggested that per 100 000 world male population, 17.1 were affected with HA compared to 1.1 affected with HB. 4 Although the clinical manifestations of both HA and HB are indistinguishable, the disease severity and the frequency of bleeding into deep tissues correlate with residual factor activity ( Figure 1). ...
... The severity of hemophilia is classified depending on the plasma level of factor activity, where the severe form is defined as a factor level less than 0.01 IU/mL (<1% of normal), moderate form as a factor level from 0.01 to 0.05 IU/mL (1%-5% of normal), and mild form with a factor level from 0.05 to 0.3 IU/mL (5%-30% of normal). 1 Severe cases of factor deficiency frequently develop spontaneous bleeding episodes that might be life-threatening with the development of intracranial hemorrhage. ...
... All negative samples for either inv-1 or inv-22 were sequenced for F8 exons 1, 3, 4, 6, 11, 12, 14, 22, and 23 with the flanking intronic regions. While for F9 gene analysis, all 39 samples of patients with HB were amplified and sequenced for all exonic regions of the gene (F9 exons 1-8) as described by Al-Allaf et al. 1 For PCR amplification, the Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany) was used. For designing the sequence of the primers, the Primer3web-based server was utilized (http://frodo.wi.mit.edu/primer3/). ...
Establishing a national screening program for hemophilia patients is highly encouraged by the World Health Organization and the World Federation of Hemophilia. Hence, this study aimed to analyze the variant spectrum of F8 and F9 genes in Arab hemophilia patients. Molecular genetic and sequencing studies were performed on a cohort of 135 Saudi hemophilia patients. Out of all screened hemophilia patients (97 hemophilia A and 39 hemophilia B), 15 (11.1%) were positive for inversion 22 and 4 (3%) for inversion 1. Out of a total of 32 (23.7%) substitution/deletion mutations, 2 novel variants were identified: a novel splice acceptor site missense mutation (c.5816-2A > G) causing a pathogenic variant of the F8 gene and another splicing site point mutation in intron/exon 23 (g.164496G > A).
... Hemophilia A (HA) and hemophilia B (HB) (OMIM: 306700 and 306900, respectively) are X-linked recessive bleeding disorders that are caused by the inheritance of genetic variants affecting F8 and F9 genes, those encoding coagulation factors VIII and IX with a consequent deficiency or dysfunction for its relevant factor. 1 The F8 gene sited at the end of the long arm of the X chromosome, at Xq28, is extremely large and structurally complex (186 932 bp; nearly 180-kb length containing 26 exons), while the F9 gene placed more toward the centromere, at Xq27.6, is considerably smaller and structurally simpler (nearly 34-kb length containing only 8 exons). 2 HA is more prevalent than HB worldwide. 3 In 2021, a meta-analysis study suggested that per 100 000 world male population, 17.1 were affected with HA compared to 1.1 affected with HB. 4 Although the clinical manifestations of both HA and HB are indistinguishable, the disease severity and the frequency of bleeding into deep tissues correlate with residual factor activity ( Figure 1). ...
... The severity of hemophilia is classified depending on the plasma level of factor activity, where the severe form is defined as a factor level less than 0.01 IU/mL (<1% of normal), moderate form as a factor level from 0.01 to 0.05 IU/mL (1%-5% of normal), and mild form with a factor level from 0.05 to 0.3 IU/mL (5%-30% of normal). 1 Severe cases of factor deficiency frequently develop spontaneous bleeding episodes that might be life-threatening with the development of intracranial hemorrhage. ...
... All negative samples for either inv-1 or inv-22 were sequenced for F8 exons 1, 3, 4, 6, 11, 12, 14, 22, and 23 with the flanking intronic regions. While for F9 gene analysis, all 39 samples of patients with HB were amplified and sequenced for all exonic regions of the gene (F9 exons 1-8) as described by Al-Allaf et al. 1 For PCR amplification, the Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany) was used. For designing the sequence of the primers, the Primer3web-based server was utilized (http://frodo.wi.mit.edu/primer3/). ...
Objective
Hemophilia A and B are X-linked recessive bleeding disorder caused by variants in the factor VIII (FVIII) and factor IX (FIX) genes. There is correlation between the type of mutation and clinical severity of these patients. Establishing national screening program for haemophilia patients is highly encouraged by the World Health Organization (WHO) and World Federation of Haemophilia (WFH). Hence we aimed to establish a genotypic data base for the nature of mutations present in Saudi population.
Case report
This retrospective descriptive study on a cohort of 136 Saudi hemophilia A and B patients.
Methodology
Molecular studies were performed to identify known and novel causative variants in hemophilia A and B families and correlated with some clinical features.
Results
There were 129 male and 7 females with age ranged from 2 - 62 years old, 97 (71.3%) hemophilia A (HA) and 39 (28.7%) hemophilia B (HB). The clinical severity of hemophilia A ranged between mild (10, 10.3%), moderate (2, 2.1%) and severe (83, 85.6%), while for hemophilia B (mild 13 (33.3%), moderate 2 (5.1%) and severe 24 (61.5%) respectively. There were 76 (55.9%) had chronic joint disability. Factor inhibitors with different titers were detected in 24 (24.7%) of HA and only 2 (5.1%) of HB. Out of the whole cohort 136 had been tested for causative variants, 17 (12.5%) were positive for inv-22 and 4 (2.9%) for inv-1, while all negative HA were selected for analysis by next generation sequencing. We are reporting 3 cases of females with severe forms of hemophilia.We are reporting different mutations that was consistent in group of tested members of same family /trip. We confirmed as previously reported high frequency of inv 22 and we found 7 novel mutation out 12 detected variants for HA and one novel mutation out of 13 detected variants for HB.
Conclusion
These results will enrich the spectrum of variants and enlarge the factor VIII and factor IX proteins database in the Saudi Arabian population. Establishing a molecular genetic based tests for fast, easy, and cost effective reliable mutation screening that can also be applied in the future for prenatal and pre-implantation genetic diagnosis.
... Both plasma-derived (pd) and recombinant clotting factor concentrates are suitable for these different strategies of hemophilia management [18]. Nowadays, many studies are being carried out on polymorphisms within the genes coding for factors VIII and IX [19] [20] [21]. Both factor VIII and IX genes contain two types of polymorphisms. ...
... PCR amplification and capillary sequencing. Amplification of the F8 gene exons was done as previously described (Al-Allaf et al., 2017a;Al-Allaf, 2017b). In summary, the gene was sequenced with 50 ng of genomic DNA as template in a 20 µl reaction mixture using 0.4 µl HotStarTaq plus DNA Polymerase (Qiagen, Hilden, Germany), 2 µl 10 × PCR buffer, 2 µl 25 mM MgCl 2 , 0.4 µl 10 mM dNTPs, 2 µl of 10 µM forward and reverse primers; descriptions of the primers used for PCR amplification and sequencing have been reported previously (Vidal et al., 2001). ...
Hemophilia A is an X-linked recessive hemorrhagic disorder caused by variants in the F8 gene. To identify known and novel causative variants in hemophilia A, we have carried out genetic analysis among Saudi patients. Twenty-one patients, who were negative for inv-1/inv-22, were selected for analysis by next generation sequencing, thereafter confirmed by Sanger sequencing. In addition, the functionality and structural changes in the variant proteins were assessed using Molecular dynamics (MD) simulation and compared with wild-type and native proteins. In the samples we analyzed, we found 10 variants in 12 individuals; among them, five were novel and five were previously reported. The novel variants were located at positions: c.6130_6131insC, c.5815G>C, c.5493C>G, c.3734_3740delinsATTTCT and c.3744A>T. With the exception of one variant which was silent, the MD simulation revealed that the observed variants were causing severe structural changes when compared to the native protein and resulted in a loss of the protein’s function. The MD analysis is in line with clinical data of patients who had <1% Factor VIII levels (severe hemophilia) with episodic bleeding, and were on more than one treatment. Moreover, some patients presented with chronic joint disability. These results will enrich the spectrum of variants and enlarge the factor VIII protein’s database in the Saudi Arabian population.
... PCR amplification and capillary sequencing. Amplification of the F8 gene exons was done as previously described (Al-Allaf et al., 2017a;Al-Allaf, 2017n). In summary, the gene was sequenced with 50 ng of genomic DNA as template in a 20 µl reaction mixture using 0.4 µl HotStarTaq plus DNA Polymerase (Qiagen, Hilden, Germany), 2 µl 10xPCR buffer, 2 µl 25 mM MgCl2, 0.4 µl 10 mM dNTPs, 2 µl of 10 µM forward and reverse primers; descriptions of the primers used for PCR amplification and sequencing have been reported previously (Vidal et al., 2001). ...
Hemophilia A is an X-linked recessive hemorrhagic disorder caused by variants in the F8 gene. To identify known and novel causative variants in hemophilia A, we have carried out genetic analysis among Saudi patients. Twenty-one patients, who were negative for inv-1/inv-22, were selected for analysis by next generation se-quencing, thereafter confirmed by Sanger sequencing. In addition, the functionality and structural changes in the variant proteins were assessed using Molecular dynamics (MD) simulation and compared with wild-type and native proteins. In the samples we analyzed, we found 10 variants in 12 individuals; among them, five were novel and five were previously reported. The novel variants were located at positions: c.6130_6131insC, c.5815G>C, c.5493C>G, c.3734_3740delinsATTTCT and c.3744A>T. With the exception of one variant which was silent, the MD simulation revealed that the observed variants were causing severe structural changes when compared to the native protein and resulted in a loss of the protein's function. The MD analysis is in line with clinical data of patients who had <1% Factor VIII levels (severe hemo-philia) with episodic bleeding, and were on more than one treatment. Moreover, some patients presented with chronic joint disability. These results will enrich the spectrum of variants and enlarge the factor VIII protein's database in the Saudi Arabian population.
تضمنت الدراسة عزل وتنقية أنزيم كلوتاثايون بيروكسيديز (GPx) في مصل دم مرضى الهيموفيليا، إذ شملت 48 عينة من مصل دم مرضى الهيموفيليا بكلا نوعيه (40 عينة من النوع A و 8 من النوع B)، علاوة على 50 عينة من الأشخاص الأصحاء ومن الذكور فقط. بينت نتائج الدراسة وجود انخفاض معنوي عند مستوى الاحتمالية P≤ 0.05 لفعالية أنزيم GPx في مصل دم مرضى الهيموفيليا بكلا نوعيه. وتم ايضا عزل وتنقية أنزيم GPx من مصل دم المصابين بالهيموفيليا من خلال الترسيب بكبريتات الامونيوم والديلزة وباستخدام تقنية كروماتوغرافيا التبادل الايوني DEAD – Cellulose حيث تم فصل حزمة بروتينية رئيسة والتي اعتمد عليها في تحديد الظروف المثلى للأنزيم المنقى جزئيا. حُددت الظروف المثلى للأنزيم المنقى جزئيا من مصل الدم وكانت أعلى فعالية لزمن التفاعل عند الدقيقة 6، دالة حامضية عند pH=8، وتركيز الانزيم 105 µg/ml، وتركيز مادة الاســــاس H2O2 L/0.6 mmol، ودرجة حرارة 40oC وان السرعة القصوى Vmax وثابت ميكيلس Km كانت µmol/min 0.9 و 0.195 mmol/Liter على التوالي باستخدام رسم لاينويفر. - برك. تضمنت الدراسة أيضا تأثير بعض النواتج الطبيعية من بذور نبات الكسوب (الزيت، الفلافونويدات، الكلايكوسيدات) على فعالية أنزيم GPx. حُددت الظروف المثلى للأنزيم المنقى جزئيا من مصل الدم وكانت أعلى فعالية عن عند الدقيقة (6)، دالة حامضية عند ( pH=8)،و تركيز الانزيم ((105 µg/ml، وتركيز مادة الاساس H2O2 (L/0.6 mmol)،و درجة حرارة(40oC) وان السرعة القصوى (Vmax) وثابت ميكيلس (Km) كانت مساوية (mol/minµ 0.9) و(0.195mmol\Liter) على التوالي باستخدام رسم لاين ويفر- برك. كما تضنت الدراسة عزل بعض النواتج الطبيعية من بذور نبات الكسوب (الزيت ، القلافونويدات ، الكلايكوسيدات) وبينت النتائج انها تزيد من فعالية الانزيم المنقى جزئيا من مصل الدم .
Remarkable changes are occurring in the diagnosis and management of individuals with hemophilia A. Genetic testing, including next-generation sequencing, enables family planning, carrier testing, and prenatal diagnosis. Musculoskeletal ultrasound examination facilitates the early detection of acute bleeds and joint disease in clinic, enabling more rapid bleed resolution and treatment planning. Novel therapies offer simpler weekly or monthly administration, some by subcutaneous injection, with better compliance and quality of life, as well as fewer bleeds. Gene therapy provides a 1-time phenotypic "cure" that is cost effective, but may be complicated by waning levels, vector immune responses, and hepatotoxicity.
Hemophilia A (HA) and hemophilia B (HB) are rare disorders, being caused by the total lack or under-expression of two factors from the coagulation cascade coded by genes of the X chromosome. Thus, in hemophilic patients, the blood does not clot properly. This results in spontaneous bleeding episodes after an injury or surgical intervention. A patient-centered regimen is considered optimal. Age, pharmacokinetics, bleeding phenotype, joint status, adherence, physical activity, personal goals are all factors that should be considered when individualizing therapy. In the past 10 years, many innovations in the diagnostic and treatment options were presented as being either approved or in development, thus helping clinicians to improve the standard-of-care for patients with hemophilia. Recombinant factors still remain the standard of care in hemophilia, however they pose a challenge to treatment adherence because they have short half-life, which where the extended half-life (EHL) factors come with the solution, increasing the half-life to 96 hours. Gene therapies have a promising future with proven beneficial effects in clinical trials. We present and critically analyze in the current manuscript the pros and cons of all the major discoveries in the diagnosis and treatment of HA and HB, as well as identify key areas of hemophilia research where improvements are needed.
By examining the issue of the thromboses and hemostasis disorders associated with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) through the lens of cross-reactivity, it was found that 60 pentapeptides are shared by SARS-CoV-2 spike glycoprotein (gp) and human proteins that— when altered, mutated, deficient or, however, improperly functioning— cause vascular diseases, thromboembolic complications, venous thrombosis, thrombocytopenia, coagulopathies, and bleeding, inter alia. The peptide commonality has a relevant immunological potential as almost all of the shared sequences are present in experimentally validated SARS-CoV-2 spike gp-derived epitopes, thus supporting the possibility of cross-reactions between the viral gp and the thromboses-related human proteins. Moreover, many of the shared peptide sequences are also present in pathogens to which individuals have previously been exposed following natural infection or vaccinal routes, and of which the immune system has stored imprint. Such an immunological memory might rapidly trigger anamnestic secondary cross-reactive responses of extreme affinity and avidity, in this way explaining the thromboembolic adverse events that can associate with SARS-CoV-2 infection or active immunization.
