Hemophilia A Carriers Research Articles

Clinical Analysis and Mental Health Survey of Hemophilia Carriers: a Cross-sectional Study.

Hemophilia carriers (HCs), who are heterozygous for mutations in the clotting factor VIII/clotting factor IX gene (F8 or F9), may have a wide range of clotting factor levels, from very low, similar to afflicted males, to the upper limit of normal, and may experience mental health issues. The purpose of this study was to provide genetic information on mothers of hemophilia patients and to understand the clotting factor activity and phenotype of HCs. Additionally, we aimed to investigate the mental health status of HCs in China. A total of 127 hemophilia mothers, including 93 hemophilia A (HA) mothers and 34 hemophilia B (HB) mothers, were enrolled in this study. Long distance PCR, multiplex PCR, and Sanger sequencing were used to analyze mutations in F8 or F9. Coagulation factor activity was detected by a one-stage clotting assay. The Symptom Checklist 90 (SCL-90, China/Mandarin version) was given to HCs at the same time to assess their mental health. A total of 90.6% of hemophilia mothers were diagnosed genetically as carriers, with inversion in intron 22 and missense mutations being the most common mutation types in HA and HB carriers, respectively. The median clotting factor level in carriers was 0.74 IU/mL (ranging from 0.09 to 1.74 IU/mL) compared with 1.49 IU/mL (ranging from 0.93 to 1.89 IU/mL) in noncarriers, of which 14.3% of HCs had clotting factor levels of 0.40 IU/mL or below. A total of 53.8% (7/13) of HA carriers with low clotting factor levels (less than 0.50 IU/mL) had a history of bleeding, while none of the HB carriers displayed a bleeding phenotype. The total mean score and the global severity index of the SCL-90 for surveyed HCs were 171.00 (±60.37) and 1.78 (±0.59), respectively. A total of 67.7% of the respondents had psychological symptoms, with obsessive-compulsive disorder being the most prevalent and severe. The pooled estimates of all nine factors were significantly higher than those in the general population (P<0.05). The detection rate of gene mutations in hemophilia mothers was 90.6%, with a median clotting factor level of 0.74 IU/mL, and 14.3% of HCs had a clotting factor level of 0.40 IU/mL or below. A history of bleeding was present in 41.2% of HCs with low clotting factor levels (less than 0.50 IU/mL). Additionally, given the fragile mental health status of HCs in China, it is critical to develop efficient strategies to improve psychological well-being.

Four Decades of Carrier Detection and Prenatal Diagnosis in Hemophilia A: Historical Overview, State of the Art and Future Directions.

Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20-22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11-14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important.

CLINICAL AND HEMORRHAGIC PROFILES OF HEMOPHILIA A FEMALE CARRIERS OF INTRON 22 INVERSION

According to the Ministry of Health, Brazil had 10,821 patients diagnosed with hemophilia A (HA) in 2019 and with Minas Gerais had the third largest population of HA patients. Fundação Hemominas is a reference treatment center in this state for the clinical and laboratory care of these patients. HA is an X-linked coagulopathy caused by a deficiency of coagulation factor VIII (FVIII). Almost all patients are male, and females are considered carriers if they have an X chromosome with a mutation in the FVIII gene. In addition, it is estimated that there are 1.6 female carriers for every male HA patient. HA Carriers are classified into five categories based on residual plasma FVIII level (FVIII:C) and phenotype: (i) FVIII:C > 40% with bleeding phenotypes are classified as symptomatic; (ii) FVIII:C > 40% without bleeding phenotypes are classified as asymptomatic; (iii) FVIII:C > 5% to 40% are classified as mild HA; (iv) FVIII:C 1% to 5% are classified as moderate HA; and (v) FVIII:C < 1% are classified as severe HA. The aim of this study was to analyze the clinical and hemorrhagic profiles of female relatives of patients with severe HA with intron-22 inversion treated at Fundação Hemominas and older than 12 years. To date, sixteen participants have been enrolled in the study after signing an informed consent form. Participants answered two questionnaires: 1) evaluation of bleeding - ISTH (BAT-ISTH); and 2) evaluation of arthralgia. Peripheral blood samples were collected in a citrate tube and processed for DNA extraction. Genotyping to identify intron 22 inversion was performed by inverse PCR. Of the 16 participants, seven (44%) were identified as carriers of the mutation. Among the carriers, the median (MED) age was 35 years (IQR 34 - 35.5) and the bleeding score MED was 6 (IQR 4.8 - 9). In addition, carriers reported pain in the joints of the shoulders (3 – 42.9%), hips (2 – 28.6%), knees (2 – 28.6%), and ankles (2 – 28.6%). For participants who were not carriers, the age MED was 36 years (IQR 30 - 56) and the bleeding score MED was 4 (IQR 1 - 7). In addition, participants who were not carriers had pain in all joints studied, i.e., shoulders (3 – 33.33%), elbows (1 – 11.11%), hips (2 – 22.22%), knees (4 – 44.44%), and ankles (2 – 22.22%). Molecular diagnosis was important to identify family members with intron-22 inversion and to provide appropriate genetic counseling for family planning. A significant statistical difference (p = 0.019) was found between carriers and non-carriers in bleeding score. This is an ongoing study and the number of participants needs to be increased in order to draw conclusions. We thank all participants, Fapemig, Fundação Hemominas, and the CGSH of the Ministry of Health.

Molecular Mechanisms of Skewed X-Chromosome Inactivation in Female Hemophilia Patients-Lessons from Wide Genome Analyses.

Introduction: Hemophilia A (HA) is an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency or dysfunction due to F8 gene mutations. HA carriers are usually asymptomatic because their FVIII levels correspond to approximately half of the concentration found in healthy individuals. However, in rare cases, a carrier may exhibit symptoms of moderate to severe HA primarily due to skewed inactivation of her non-hemophilic X chromosome. Aim: The aim of the study was to investigate X-chromosome inactivation (XCI) patterns in HA carriers, with special emphasis on three karyotypically normal HA carriers presenting with moderate to severe HA phenotype due to skewed XCI, in an attempt to elucidate the molecular mechanism underlying skewed XCI in these symptomatic HA carriers. The study was based on the hypothesis that the presence of a pathogenic mutation on the non-hemophilic X chromosome is the cause of extreme inactivation of that X chromosome. Methods: XCI patterns were studied by PCR analysis of the CAG repeat region in the HUMARA gene. HA carriers that demonstrated skewed XCI were further studied by whole-exome sequencing (WES) followed by X chromosome-targeted bioinformatic analysis. Results: All three HA carriers presenting with the moderate to severe HA phenotype due to skewed XCI were found to carry pathogenic mutations on their non-hemophilic X chromosomes. Patient 1 was diagnosed with a frameshift mutation in the PGK1 gene that was associated with familial XCI skewing in three generations. Patient 2 was diagnosed with a missense mutation in the SYTL4 gene that was associated with familial XCI skewing in two generations. Patient 3 was diagnosed with a nonsense mutation in the NKAP gene that was associated with familial XCI skewing in two generations. Conclusion: Our results indicate that the main reason for skewed XCI in our female HA patients was negative selection against cells with a disadvantage caused by an additional deleterious mutation on the silenced X chromosome, thus complicating the phenotype of a monogenic X-linked disease. Based on our study, we are currently offering the X inactivation test to symptomatic hemophilia carriers and plan to expand this approach to symptomatic carriers of other X-linked diseases, which can be further used in pregnancy planning.

Random X-Chromosome Inactivation in Hemophilia A Carriers Does Not Fully Attenuate Bleeding Risk

Background: In contrast to males with the X-linked bleeding disorder Hemophilia A (HA), bleeding in female carriers is attenuated by the presence of a wild type F8 allele. While low FVIII activity (FVIII:C) is more predictive of bleeding tendency, hemophilia carriers with “normal” FVIII:C can have bleeding sequelae. FVIII:C in HA carriers is influenced by X-chromosome inactivation (XCI), a process that silences one X in XX females. XCI results in cells that have randomly inactivated either parental X, although skewed XCI patterns occur commonly in ~25% of the general population. How frequently XCI skewing in HA carriers leads to preferential activation of the X carrying the F8 mutation, increasing bleeding tendency, is poorly understood. We sought to determine whether XCI skewing further differentiates bleeding risk in HA carriers.Methods: Patients were enrolled in this prospective, observational cross-sectional study with informed consent when presenting for F8 genotype analysis through the My Life, Our Future (MLOF) study. Standardized Condensed MCMDM-1 and Pediatric Bleeding Questionnaire (PBQ) bleeding assessment tools (BAT) and Pictorial Blood-loss Assessment Charts (PBAC) were completed at the time of the visit. Hemophilia carrier status was confirmed by documented F8 mutation. One-stage FVIII:C assays and chromogenic FVIII assays were performed on all subjects. XCI skewing was assessed using established assays at the Androgen Receptor (AR) and Fragile X Mental Retardation (FMR1) loci.Results: 98 individuals have been evaluated to date. 65 (46 adults, 19 adolescents and children) are confirmed F8 mutation carriers, while 33 (17 adults) are confirmed non-carriers. 39 (30 adults) have F8 mutations associated with severe HA, and 26 (16 adults) have non-severe F8 mutations. 21/46 (46%) adult HA carriers have BAT scores >4. BAT scores are significantly higher (5 vs. 1.8, p=0.0452) in adult HA carriers compared to age-matched controls (39.9 vs. 35.5, p=0.24). PBAC and BAT correlate well in adult F8 carriers (r =0.62, p<0.0001). Among adolescents, BAT scores are similar between controls and carriers; none of the HA carriers have BAT scores >2.FVIII assessment by one-stage and chromogenic FVIII assays are highly correlated (p<0.0001), with the notable exception of one carrier whose assays are markedly discrepant (19% chromogenic vs 108% one-stage). Her F8 mutation (p.Glu1988Argfs*4), is associated with severe HA and maps within the A3 domain. Although mutations in this domain can result in discrepant one-stage/chromogenic activity, such an association has not been previously reported for this specific mutation. As chromogenic results more accurately reflect bleeding tendency in this carrier, utilization of both activity assays will capture more individuals at potential risk.Notably, only one third (7/21) of adult HA carriers with significant bleeding have FVIII:C below 40%. 4/21 have FVIII:C in the 40-60% range, and 10/21 have “normal” levels >60%, indicating that FVIII:C levels alone cannot predict bleeding risk in all carriers. Indeed, for all adult HA carriers (n=46), chromogenic and one-stage FVIII assays correlate only weakly with BAT (r=-0.29, p=0.053) and PBAC (r=-0.27, p=0.072) scores. However, the correlation for severe F8 mutation carriers (n=30) is stronger between BAT scores and chromogenic assays (r=-0.37, p=0.0464), and PBAC scores and one-stage assays (r=-0.36, p=0.0493).XCI assays are informative in 44 of 46 adult HA carriers. 21/44 adult carriers have skewed XCI (>70:30) and 9 are extremely XCI skewed (>80:20). This level of XCI skewing exceeds that in the general population. Although random XCI is frequently proposed to attenuate carrier bleeding phenotype, 9/23 HA carriers with random XCI have BAT scores >4, including 2 with non-severe F8 mutations predicted to have a mild phenotype. Adolescent HA carriers have similar proportions of XCI skewing and similar FVIII activity to adult HA carriers, yet their reported bleeding is indistinguishable from age matched controls.Conclusion: Contrary to current dogma, bleeding tendency in HA carriers is not rare, and random X-chromosome inactivation is not fully protective. Correlation of bleeding scores and FVIII:C in adult HA carriers is strengthened when stratified by F8 mutation severity. Additional factors must influence hemostasis for HA carriers with random XCI. DisclosuresCygan:Hemostasis and Thrombosis Research Society: Research Funding.

Genetic analysis for carrier diagnosis in hemophilia A and B in the Mexican population: 25 years of experience.

Our 25 years of experience in carrier diagnosis of hemophilia A (HA) and B (HB) in Mexican population comprises linkage analysis of intragenic F8/F9 neutral variants along with, in severe HA (SHA), detection of F8 int22h and int1h inversions. In symptomatic carriers (SCs) we explored Lyonization to explain their symtomatology. From a DNA-Bank of 3,000 samples, intragenic restriction fragment length (RFLPs) and short tandem repeats (STRs) of F8/F9 genes were assessed by PCR-PAGE and GeneScan. In SHA patients, F8 inversions were detected by inverse shifting-PCR/diagnostic and complementary tests. In SCs, we evaluated hemorrhagic symptoms, clotting FVIII/FIX and X-chromosome inactivation (XCI) patterns were assessed by HUMARA assay and the search of XIST promoter pathogenic variants. Informativeness of linkage analysis for HA carrier diagnosis with RFLP's/STR's increased to 74% and reached 80% with five RFLPs for HB. Combined Inv22/Inv1 diagnosed 113 possible carriers, three de novo Inv22-1, and confirmed 45 mothers as obligate or sporadic carriers. Among 21 SCs, four showed extreme skewed XCI pattern (~80:20) but had normal karyotype and no C43G pathogenic variant in XIST promoter. Clotting FVIII/FIX correlated with the active X in leukocytes. Our data integrate the largest comprehensive research worldwide on the molecular diagnosis of HA and HB carriers in terms of the number of studied and diagnosed cases, in addition to the genetic analysis in SCs. Intragenic RFLPs and STRs of F8/F9 genes along with F8 int22h/int1h inversions in SHA emerge as optimal variants for molecular diagnosis in Mexican population. In counseling SCs, inheritance of skewed X-inactivation should be considered.

Evaluation of Maternal Safety Following Prenatal Cell and Gene Therapy for Hemophilia a in a Large Animal Model Demonstrates Absence of Maternal Exposure to the Cells or Gene Products Infused into the Fetus

Prenatal transplantation (PNT) using stem/progenitor cells has been performed in more than 50 patients using an ultrasound-guided approach, proving that this procedure poses minimal risk to both the fetus and the mother. Since 75% of hemophilia A (HA) patients have a family history of HA, and prenatal diagnosis is available to pregnant HA carriers in most western countries, PNT constitutes a clinically viable approach to treat these patients. In addition, the type of mutation and family history can predict the severity of the disease and the patient's likelihood of developing FVIII inhibitors, thus allowing intervention in those who could most benefit from this approach. Nevertheless, maternal safety is a critical consideration for PNT, particularly with respect to possible exposure to the cells or gene products produced by the cells infused into the fetus. We have previously demonstrated that transplanting sheep fetuses (n=23) with human placental cells (PLC) transduced with a lentiviral vector encoding a bioengineered fVIII transgene (mcoET3), at a dose of 107-108 cells/kg, increased plasma fVIII activity levels for at least 3 years after transplant. Therefore, here we studied ewes (n=4) during pregnancy and those who had given birth (n=6) to lambs treated in utero. The latter were assessed longitudinally over a course of 5 months, ranging from 18 to 32 months after delivery of a PNT recipient. First, we investigated whether any of the ewes, during pregnancy or at any point thereafter,developed an immune response to the transplanted product, PLC-mcoET3. Peripheral blood mononuclear cells (PBMC) harvested from the different ewes readily proliferated in response to human PBMC and upon PHA stimulation, but PLC-mcoET3 and non-transduced PLCs did not elicit an immune response from PBMC from any of the ewes, demonstrating that PNT did not immunize the ewe to the transplanted cells. We also used an mcoET3-specific ELISA to investigate whether the ewes developed antibodies to the mcoET3 protein. At no time point were ewes ever found to harbor anti-mcoET3 IgGs in their plasma. To determine whether mcoET3-specific T effector cells developed in the ewes, we performed ELISpot assays using PBMC collected during pregnancy and at various time points thereafter, and no mcoET3-specific Th1 or Th2 cells were detected in these ewes. To assess the potential circulation and/or engraftment of PLC-mcoET3 in the ewes during pregnancy or at any time after delivery, maternal PBMC collected during pregnancy, and maternal-side placental tissue and umbilical cord tissue (UBC) collected at birth were analyzed using RT-qPCR (detection limit &amp;lt;0.01%) with primers specific for human FVIII (produced constitutively by the PLC) and mcoET3. Maternal PBMC, UBC, and placental tissue were all devoid of PLC-mcoET3, as evidenced by the complete absence of mcoET3 and human FVIII mRNA. Histopathologic analysis of the placental tissue demonstrated no abnormalities or ectopic tissue formation. Overall, these studies suggest that the administration of PLC-mcoET3 to the fetus during gestation does not result in detectable maternal exposure to the cells or gene products, attesting to the safety of this approach. Disclosures Doering: Kilpatrick, Townsend &amp; Stockton: Consultancy; Expression Therapeutics, LLC: Current equity holder in private company, Patents &amp; Royalties, Research Funding.

Application of Indirect Linkage Analysis for Carrier Detection of Hemophilia A in Kurdistan Region of Iraq: Usefulness of Intron 18 BclI T>A, Intron 19 HindIII C>T, and IVS7 nt27 G>A Markers.

Hemophilia A (HA) is the most common congenital X-linked coagulopathy caused by mutations in the factor VIII gene. One in 5000 to 10 000 male persons worldwide suffer from HA. It is the archetype of high-cost, low-volume disease. Therefore, identification of carriers is crucial to avoid the birth of affected males. Tracking of the defective X chromosome through indirect linkage analysis represents the most practical method for screening for carriers in developing countries. In this study, 227 individuals from 41 families with HA and 100 normal participants were recruited from the Kurdistan region of Iraq and evaluated for intron 18 BclI, intron 19 HindIII, and IVS7 nt 27 markers by polymerase chain reaction restriction fragment length polymorphism and direct sequencing. Among the studied women, 49%, 42%, and 14% were discovered to be heterozygous for BclI, HindIII, and IVS7 markers, respectively. Using BclI, HindIII, and IVS7 markers, 56%, 46%, and 17% of the families were informative, respectively. The combined informativity of these polymorphic sites reaches 66%. The current study illustrates the effectiveness of the BclI and HindIII markers for the diagnosis of HA carriers among the Iraqi Kurdish population.

Comprehensive Evaluation of Bleeding Disorders in Adolescents with Heavy Menstrual Bleeding

Introduction: Unpredictable, prolonged or heavy menstrual bleeding (HMB) is not unusual in many adolescents soon after menarche. Anovulation, owing to immaturity of the hypothalamic-pituitary-ovarian axis, is often the cause of HMB; however, it may coexist and mask an underlying bleeding disorder (BD). Retrospective data in adolescents with HMB suggest a prevalence of BDs from 10 to 62%. Prospective studies with uniform hemostatic evaluation and concurrent quality of life (QoL) assessments have not been performed. The incidence of BDs in adolescents, especially those with anovulatory bleeding, remains unknown. Methods: In this ongoing prospective cohort study (targeted n=200), 130 adolescents referred for HMB to multidisciplinary Young Women's Blood Disorders Clinics were enrolled. An assessment of menstrual loss using pictorial blood assessment chart (PBAC) and bleeding symptoms using ISTH-bleeding assessment tool (BAT) was undertaken. Participants underwent a comprehensive evaluation determined a priori, including assessment of hypermobility, standardized laboratory evaluation (prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, von Willebrand disease (VWD) panel, whole blood platelet aggregation with ATP secretion, factor assays (FIX and FXIII), global hemostasis and hyperfibrinolysis using ROTEM, and a pelvic ultrasound (US). Patient reported outcomes for health related QoL(HR-QoL) was completed. Results: The mean age of participants was 14 (range 11-18) and mean age at menarche was 11.7 yrs. (range: 9-16). The mean PBAC score, before contraceptive initiation, was 504 (range: 100- 1200) and the mean ISTH BAT score was 3.8 (range: 1-8) in all participants. 62% had anovulatory menstrual bleeding. An inherited BD and/or bleeding risk was diagnosed in 24.6% (n=32); 9% (n=12) were diagnosed with VWD, 5% (n=6) with qualitative platelet dysfunction and 2 subjects were found to be hemophilia A (HA) carriers. Low VWF levels (VWF:Rco 30-50%) were detected in 9% (n=12). 15% had evidence of mild systemic hyperfibrinolysis (lysis 3-8% on ROTEM) but none met the criteria for further investigation for an inherited disorder of hyperfibrinolysis . Of those without BD, 2 were diagnosed to have VWF exon 28 polymorphism, and 3 were referred and ultimately diagnosed with hypermobility syndrome by Genetics. One was found to have endometriosis by laparoscopy and 3 were diagnosed with PCOS. Of those with BDs (all with low VWF), 3 were diagnosed with hypermobility syndrome (total 6) and 4 with PCOS (total 7). US was normal in all subjects. The PBAC scores were higher in BDs (634 vs. 410; SD ± 275 p=0.002), irrespective of the pattern of bleeding. No differences were found in the ISTH BAT scores (after excluding HA carriers) (3.6 vs. 4.8; SD ± 1.19, p=0.06), initial hbg or ferritin levels between the two groups. Only 6% (n=8) with BDs required ED visit and/or hospitalization for HMB vs. 14% (n=18) without a BD, of which majority had anovulatory bleeding. On multivariate logistic regression, no predictors of BDs were found. Adolescents with BDs had a decreased HR-QoL compared to those without a BD (QoL score 58 vs.87; SD± 12.5, p=&lt;0.001) even after control of HMB and correction of anemia. Conclusions: Inherited BDs are common in adolescents with HMB, confer a decreased HR-QoL and can be associated with anovulatory bleeding. A comprehensive evaluation is required to unravel disorders causing HMB. In this ongoing study, at this time, no predictor of an inherited BD was found in adolescents with HMB. Data accrual from the fully powered, ongoing, prospective study may offer further data to develop an ideal tool with a scoring system to take into account all prevalent causes of HMB to accurately predict a BD in adolescents. Disclosures Zia: NHLBI K23: Research Funding. Journeycake:CSL: Consultancy; Biogen: Consultancy; Baxalta/Shire: Consultancy. Jain:Novo Nordisk: Honoraria; Bayer: Membership on an entity's Board of Directors or advisory committees; Biogen: Speakers Bureau. Sarode:CSL Behring: Consultancy, Honoraria.

Genetic diagnosis in Hemophilia A from southern China: five novel mutations and one preimplantation genetic analysis.

As there is currently no complete cure for hemophilia A (HA), the identification of pathogenic mutations in factor VIII (FVIII) gene from HA patients and carriers, which can contribute to genetic counseling prenatal diagnosis, and preimplantation genetic diagnosis (PGD), is an important step to prevent HA. A total of 14 unrelated Chinese HA subjects (FVIII activity <40%), 20 carrier subjects, three fetuses, and one PGD were included in this study. We first screened for the presence of FVIII intron 22 and intron 1 inversions. Second, the coding region of the FVIII gene was sequenced. For the novel mutations, FVIII mRNA expression was detected by real-time PCR and the protein structures were analyzed by bioinformatic tools. Five novel mutations (c.1A>C, c.304_305insA, c.1594T>A, c.6045G>A, and c.2645_2646insG) were found. The real-time PCR showed that the expression of FVIII mRNAs was lower in HA patients than in normal subjects. Prenatal diagnosis and PGD were successfully performed: Two of three fetuses and four of eight blastomeres were confirmed to be normal. In conclusion, genetic diagnosis of 14 unrelated HA subjects, 20 carrier subjects, three fetuses, and one PGD was successfully performed in our study.

Evaluation of factor VIII polymorphic short tandem repeat markers in linkage analysis for carrier diagnosis of hemophilia A.

Hemophilia A (HA) is the most common inherited X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene (FVIII). Diagnosis of the carrier is critical for preventing the birth of children affected by this coagulation disorder, which ultimately facilitates its management. Due to the heterogeneous nature of mutations, the large inversions and the complexity of the FVIII gene, direct recognition of the disease-associated mutation in HA is complex. Indirect linkage analysis using highly informative heterozygous polymorphic markers is an alternative method for determining the co-segregation of the mutant gene within a family for carrier detection of HA. The aim of the present study was to perform carrier diagnosis in a family with HA. Rapid multifluorescent polymerase chain reaction (PCR) was performed with six extragenic short tandem repeats (STRs), DXS1073, DXS15, DXS8091, DXS1227, DXS991, DXS993 and one intragenic marker, STR22 for linkage analysis in the HA family. All the STR markers employed in the present study were informative for linkages of pathogenic and healthy haplotypes among family members, particularly STR22, DXS1073 and DXS15. The STR marker, STR22, is within the FVIII gene while the DXS1073 and DXS15 markers are very close to the FVIII gene, where the chances of recombination are comparatively low, and provided the most accurate interpretation analysis, indicating that the proband's sister may have been the HA carrier. Rapid multifluorescent PCR using STR markers and linkage analysis was identified to be a simple method for performing HA carrier diagnosis.

Genotyping of Intron Inversions and Point Mutations in Exon 14 of the FVIII Gene in Iranian Azeri Turkish Families with Hemophilia A.

Hemophilia A (HA) is an inherited X-linked bleeding disorder caused by a variety of mutations that are distributed throughout the large FVIII gene (F8). The most common mutations in studied populations with severe HA are introns 22 and 1 inversions, gross exon deletions and point mutations in exon 14. The aim of this study was to define the frequency of these common mutations in Iranian population of Azeri Turkish in North West of Iran. Fifty patients with severe HA and forty-three female potential carriers were genotyped by inverse shifting polymerase chain reaction (IS-PCR), long-range PCR, multiplex PCR, and sequencing methods for the detection of Intron 22 and 1 inversions, gross exon deletions, and exon 14 point mutations, respectively. F8 intron 22 inversion was detected in 22 (44%) out of 50 patients. Moreover, we detected one intron 1 inversion (2%), and one point mutation in exon 14 (2%). In this population, 52% of the patients with hemophilia A did not show to carry a mutation in the analyzed regions by three mentioned methods. F8 intron 22 inversion was the major causative mutation in nearly 50% of severe HA cases in an Azerbaijani Turkish population, which is similar to the incidence of other populations. IS-PCR is a robust, rapid, efficient, and cost-effective method for the genetic analysis of patients with severe HA and for HA carrier detection, especially in developing countries.

Badania genetyczne w diagnostyce hemofilii B

Hemophilia A (HA) is one of the most frequently recognized genetically determined bleeding disorders. At its ground is a congenital lack, deficiency or dysfunction of coagulation factor VIII (FVIII), which leads to significant disturbances of hemostatic system. The severity of bleeding diathesis is determined by the degree of FVIII deficiency in plasma. The diagnosis of HA is mainly based on the results of the coagulation plasma test, including measuring the FVIII coagulation activity. Nowadays, it is believed that diagnosis of HA should be extended to genetic testing, to get to know the causative mutation. Determination of the mutation responsible for HA is important for at least two reasons: 1) enables a preliminary assessment of the risk of FVIII inhibitor development and 2) make possible the clear-cut diagnosis of HA carrier status. This paper presents an algorithm forgenetic testing in patients with HA. On top of that characterizes the molecular techniques used indiagnostic process, and discusses the difficulties associated with the interpretation of the results.

Correlation of X Chromosome Inactivation Skewing and Bleeding Phenotype in Obligate Carriers of Hemophilia A

Background: Hemophilia A (HA) is an X-linked recessive disorder that affects males, whereas female carriers are presumed asymptomatic if Factor VIII activity levels (FVIII:C) fall within normal range. However, FVIII:C does not always correlate with bleeding phenotype, leading to an underappreciation of bleeding sequelae in females. Therefore, it is clinically important to identify HA carriers at higher bleeding risk. FVIII expression in HA carriers is influenced by X chromosome inactivation (XCI), a process that silences one X in XX females such that each cell has a random probability of inactivating either parental X. However, rare female carriers of X-linked disorders can be severely affected if XCI is skewed and the mutant X is preferentially active. How XCI skewing modulates bleeding in mild/moderate HA is less well understood. HA bleeding may be also affected by variants in factors influencing FVIII binding and clearance, including Von Willebrand Factor (VWF) and ABO blood type. To better understand HA carrier bleeding tendency, we analyzed a family that segregates a mild/moderate HA mutation (F8: c.2167G>A). Four carriers in this pedigree have FVIII:C that approach affected males, necessitating prophylaxis prior to surgical procedures. We hypothesized that bleeding in these carriers can be largely explained by XCI skewing, but additional variants may fine tune FVIII:C.Methodology: FVIII levels were assessed by one stage (FVIII:C) and chromogenic (FVIII:CR) assays. At least two plasma samples spanning >3 years from each female were tested in duplicate with each FVIII assay. To address XCI skewing, we performed methylation-based assays at the Androgen Receptor (AR) and Fragile X Mental Retardation 1 (FMR1) loci. At least three independent PBMC DNA samples from each female were evaluated. Additionally, we screened VWF regions known to influence FVIII:C (exons 18-20, 24-27).Results: For each female, results between XCI assays were indistinguishable (r2 = 0.99). Two of four females had pronounced skewing (≥80:20); a third had measurable skewing (67:33). Importantly, all three predominantly expressed the mutant paternal allele. Familial XCI skewing argues for a genetic cause. However, XIST, the major regulator of XCI, lacked promoter alterations. Importantly, there was linear correlation between XCI skewing and FVIII:C measured by FVIII:C or FVIII:CR assays (r2 = 0.77 and 0.83, respectively). One female with random XCI, had FVIII:C considered hemostatic (median 51%, range 43-67), whereas the other females with skewed XCI had levels <40% (16%, range 14-20, 28%, range 26-32, and 30%, range 23-35). Notably, two females had similar FVIII:C (30% and 28%) but a greater XCI skewing discrepancy (80:20 vs. 67:33). While these two females were heterozygotes for VWF p.Thr789Ala, reported to be associated with 7% higher FVIII:C, neither ABO blood type nor any additional VWF variants known to affect FVIII binding differentiated these two individuals. Therefore, it is likely that XCI skewing primarily explains their bleeding tendency.Conclusions: Our results indicate that HA carrier bleeding phenotypes are multifaceted, and the major determinant of FVIII:C is XCI. These results also suggest that even moderate XCI skewing could influence clinical bleeding in HA carriers. However, random XCI in one female explains FVIII:C but does not fully negate bleeding tendency, emphasizing the complexity of carrier phenotype. These findings provide justification for an expanded study of carriers in unrelated pedigrees using a comprehensive approach to include XCI assays. DisclosuresNo relevant conflicts of interest to declare.

Role of Von Willebrand Factor in Female Carriers from an Extended Family with Mild Hemophilia A

Background: Hemophilia A (HA) is an X-linked disorder that primarily affects males, although female carriers can exhibit bleeding phenotypes. Factor VIII activity levels (FVIII:C) in XX females are influenced by X chromosome inactivation (XCI), a process that silences each parental X in a proportion of cells. XCI skewing can decrease FVIII expression by preferentially inactivating the normal X chromosome. FVIII:C is further modulated by factors such as ABO blood groups and von Willebrand factor (VWF). The D'/D3 domain of VWF binds circulating FVIII protein, preventing proteolytic degradation. In type 2 Normandy von Willebrand disease (2N VWD), D'/D3 mutations decrease affinity to FVIII and result in bleeding events similar to mild/moderate HA. Variants in VWF also affect the pharmacokinetics of recombinant FVIII. Current clinical screening tests detect the 3 VWF mutations responsible for >90% of 2N, but report variants not directly responsible for 2N as clinically benign. However, common polymorphisms are known to affect FVIII:C in normal individuals. Therefore, a better understanding of how specific alterations in VWF modulate HA phenotype is necessary to interpret clinical presentation and refine management with factor concentrates. This is particularly important in HA individuals carrying mild/moderate mutations. To evaluate VWF variants in HA carriers, we focused on an extended pedigree that includes four obligate carriers from a family with mild/moderate HA (FVIII: p.Ala723Thr). FVIII:C varied and largely correlated with XCI skewing. Nevertheless, the FVIII:VWF interaction prompted us to identify VWF gene variants that could further modulate FVIII:C and contribute to bleeding in this family.Methods: To identify D'/D3 VWF variants that impact FVIII binding, primers were designed to amplify exons 17-20 and 24-27 on chromosome 12. A chromosome 22 pseudogene, with 97% identity to VWF exons 24-34, complicated primer design. Primers specific to VWF were selected by targeting regions that differed from the pseudogene and were verified by digestion with a restriction enzyme unique to each chromosome 12 exon. The PCR products were amplified and sequenced from the four female carriers and a control male relative.Results: After excluding the three most common mutations responsible for 2N, seven other variants were identified. Four of these were intronic polymorphisms and a synonymous variant at p.Asn1138 not associated with VWD and presumed to be clinically benign. All but one of these have been described in normal individuals. Two females were heterozygous for missense variant rs1063856 (p.Thr789Ala) and synonymous polymorphism rs1063857 (p.Tyr795=) that are in linkage disequilibrium and are likely to impact FVIII:C and VWF antigen (VWF:Ag) levels. These common variants, found in up to 36% of Caucasians and 58% of African Americans, are reported to increase VWF:Ag and FVIII:C jn heterozygotes (9 IU/dL and 7 IU/dL respectively). Neither ABO blood groups nor XCI skewing could fully explain the differences in FVIII:C activity observed with this variant.Conclusions: We propose that VWF variants rs1063856/rs1063857 may contribute to FVIII:C differences between two females in the pedigree with similar XCI skewing. We conclude that consideration of VWF variants is important for fully understanding bleeding phenotype and treatment responses in female carriers and males in families with mild/moderate HA. These findings support the need for expanded studies into the role of FVIII and VWF variant interactions in additional unrelated HA individuals. DisclosuresNo relevant conflicts of interest to declare.

Phenotype–genotype correlations in hemophilia A carriers are consistent with the binary role of the phase between F8 and X‐chromosome inactivation

The recessive X-linked disorder hemophiliaA (HA) is rarely expressed in female carriers, most of whom express about half of normal factorVIII activity ( C). To propose an integrative assessment model for the binary role of the phase between the mutated F8 and the active X-chromosome (Xa) in C in HA carriers. We studied 67 females at risk of severe HA, comprising five symptomatic females ( C<1.5IUdL(-1) ) and 14 controls. A correlation study between C (observed vs. expected) and X-chromosome inactivation (XCI) patterns (XIPs; androgen receptor gene [AR] system) in blood leukocyte DNA was performed in carriers, by comparison of a model correlating C and XIP with arbitrary models devoid of biological significance, and with C levels in non-carriers (mean model) as a proxy from background data dispersion not influenced by XIP. We provide proof-of-concept example from a family presenting with extremely skewed XIPs in which the severe HA phenotype appeared in a heterozygous carrier of a crossover between AR and F8 loci that phased the mutated F8 with the maternally inherited Xa. Furthermore, four cases of severe HA affected women who had a combination of a heterozygous F8 mutation and extremely skewed XIPs in leukocytes or oral mucosa are presented. Correlation analyses between C levels and XIPs in carriers (n=38) but not in non-carriers (n=20) showed highly significant differences between the proposed correlation model and models without biological significance. The data support a binary influence of XCI, either increasing or decreasing the C, subject to the underlying phase set between the F8 mutation and XCI. Our evidence suggests that the phase between XCI and mutated F8 acts as a molecular switch conditioning C levels and HA expression in carriers.

Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis

An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45–50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512bp were recognized for wild type; 333, 457 and 584bp for Inv22-1; 385, 405 and 584bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333bp for Inv22-1 carrier and 385bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5min. The separation voltage was set at 8kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89mM Tris, 89mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1μM of YO-PRO®-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR.

Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverse-shifting polymerase chain reaction and capillary electrophoresis.

This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40-50% of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris-borate-EDTA (TBE) buffer containing 0.3% (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10kV for 10s at a temperature of 25°C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1%). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.

Detection of Hemophilia A Carriers in Azeri Turkish Population of Iran

Hemophilia A (HA) is an inherited X-linked coagulation disorder caused by the deficiency of factor VIII (FVIII). Linkage analysis is a common indirect method for the detection of female carriers in families with HA. In the current study, 173 patients from 30 unrelated families with HA were recruited from the Azeri Turkish population of northwest Iran and analyzed for BclI and HindIII markers by polymerase chain reaction-restriction fragment length polymorphism. We investigated the potential of using these markers for the detection of mutation in carriers through linkage analysis, which would be of tremendous use in prenatal diagnosis. Among the tested women, 47% and 35% were found to be heterozygous for BclI and HindIII polymorphic markers, respectively. The BclI and HindIII markers were informative for the detection of 63% and 17% potential carriers, respectively, demonstrating the effectiveness of the BclI marker for the detection of HA carriers among the Azeri Turkish population.

2 ANALYSIS OF METHYLATION PATTERN IN FEMALES WITH LOW FACTOR VIII: C LEVEL

The analysis of X chromosome activation through the metilation pattern could be useful in the study of affected females for X-linked recessive diseases, such as Hemophilia A (HA). Low FVIII;C level in female can be due to: acquired inhibitors to FVIII, X chromosome abnormalities, homozygosity at the hemophilia locus, extreme lyonization in an heterozygote. It is possible to reveal a non random inactivation of X chromosomes by hybridization of genomic DNA with M27ß probe (locus DXS 255). We analyzed with this probe 3 females with low FVIII:C levels (range 2%-5%). G.V. is a 33 year old female (FVIIhC 5%, FVIIIvW:Ag 80%) born from a HA carrier and a normal man. Karyotype was 46,XX and no inhibitors against FVIII were detected. The study with M27ß probe showed that only the paternal X chromosome was inactive. HA in this female is probably due to a non random X-inactivation. P.M. is a 48 year old female (FVIII:C 12%; FVIIIvW:Ag 100%) born from first cousins parents. Her father and brother are hemophilics (FVIII:C 11%) and her mother was a carrier. Cytogenetic studies revealed no abnormalities. M27ß analysis showed a balanced lyonization. An homozygosity for the HA defective gene, was demonstrated by the aplotype analysis with RFLPs. P.T. is a 19 year old female (FVIII:C 2.2%; FVIIIvW:Ag 73%) with Turner Syndrome (46,X,-idic(X)) born from healthy unrelated parents without history of bleeding disorders. The RFLP studies showed the maternal origin of idic(X). The methylation pattern obtained by M27ß probe confirmed the whole inactivation of the abnormal X therefore we hypothesized a mutation in FVIII gene in the normal X coming from her healthy father. In conclusion, the possibility to demonstrate the degree of X-chromosome activation is a very important and useful tool in understanding the pattern of HA carriers.