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ABSTRACT: We have identified 1,135 haemophilia A patients with missense mutations associated with mild (46%), moderate (22%), severe (16%), and mixed haemophilia phenotypes (11%). Altogether, we detected 374 different missense mutations of which 195 are not listed in the HAMSTeRS database. While missense mutations are strongly underrepresented within the factor VIII (FVIII) B-domain, they are evenly distributed throughout the entire F8 cDNA sequence. Only 36 (5%) of 720 patients with missense mutations and known inhibitor status showed an association with inhibitor formation. Inhibitor prevalence was four-fold higher for severe haemophilia compared to mild/moderate phenotypes. Mutations associated with inhibitor formation were especially clustered within the C1/C2 domain compared to the other domains (8.7% C1/C2 domain vs. 3.6% non-C1/C2-domain; p-value: 0.01). Three different missense mutations (T314A [T295A], S2010 [S1991P], R2169H [R2150H]) were associated twice with inhibitor formation. Importantly, we found that the risk of inhibitor formation in association with FVIII missense mutations is significant higher if the amino acid substitution belongs to another physicochemical class than the original residue (p-value 0.039). For this purpose distinct classes of substitutions were grouped in association with side chains properties: class I, small/hydrophobic; class II, neutral; class III, acidic; class IV, basic. Thus, although missense mutations were associated with an overall lower risk of inhibitor formation compared to other F8 gene mutation types, different missense mutations correlate with specific risks for inhibitor formation. These differences have to be identified in assigning risk profiles to aid in choice of preventative treatments designed to prevent inhibitor formation.
Thrombosis and Haemostasis 01/2013; 109(3). · 5.04 Impact Factor
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ABSTRACT: Recombinant factor VIII (rFVIII) concentrates differ due to cell lines, culture conditions, presence of the B domain and authorized potency assays. This study characterizes three commercially available rFVIII concentrates: a second-generation full length (A), a third-generation full length (B) and a third-generation B domain-deleted (BDD) product (C). rFVIII concentrates were characterized for FVIII activity (FVIII:C) by one-stage clotting and chromogenic assays, FVIII antigen (FVIII:Ag), thrombin activation profile and FXa-generation assay. The rFVIII concentrates exhibited significant differences with regard to FVIII:C, FVIII:Ag and thrombin activation profile. Product A had significantly greater FVIII:C and FVIII:Ag relative to the measured values of products B and C. In addition, product A demonstrated faster and more complete activation by thrombin than the two others. BDD product C had the slowest measured thrombin activation rate. Product A exhibited a greater in vitro FXa generation than products B and C. We found no differences in FXa generation among all three products when FXa generation was normalized for FVIII:Ag. The greater FVIII:C and FVIII:Ag values for product A compared with that for products B and C are due to application of different authorized potency assays (one-stage assay for A vs. chromogenic assay for B and C). The variation in thrombin activation profiles may arise from differences in cell line-dependent posttranslational modifications of the various recombinant proteins.
Haemophilia 12/2012; · 2.60 Impact Factor
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ABSTRACT: Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of the von Willebrand factor (VWF). VWD is classified into three types - type 1 (partial quantitative deficiencies), type 2 (qualitative defects) and type 3 (complete deficiency of VWF). In this study we explored genotype and phenotype characteristics of patients with VWD with the aim of dissecting the distribution of mutations in different types of VWD. One hundred fourteen patients belonging to 78 families diagnosed to have VWD were studied. Mutation analysis was performed by direct sequencing of the VWF . Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The impact of novel candidate missense mutations and potential splice site mutations was predicted by in silico assessments. We identified mutations in 66 index patients (IPs) (84.6%). Mutation detection rate was 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected comprising 37 missense mutations (54.4%), 10 small deletions (14.7%), two small insertions (2.9%), seven nonsense mutations (10.3%), five splice-site mutations (7.4%), six large deletions (8.8%) and one silent mutation (1.5%). Twenty-six of these mutations were novel. Furthermore, in type 1 and type 2 VWD, the majority of identified mutations (74% vs. 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). Genotyping in VWD is a helpful tool to further elucidate the pathogenesis of VWD and to establish the relationship between genotype and phenotype.
Thrombosis and Haemostasis 08/2012; 108(4):662-71. · 5.04 Impact Factor
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ABSTRACT: Deficiencies of natural anticoagulant proteins including antithrombin (AT), protein C (PC) and protein S (PS) are important causes of inherited thrombophilia. This study aimed to report on the practical experience gained in performing genetic analyses of a large cohort of patients with AT, PC and PS deficiencies and to relate this knowledge to clinical application. We genotyped a large cohort of 709 unrelated patients with AT (231), PC (234) and PS (244) deficiencies referred to us by physicians throughout Germany. Mutations were detected by direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. Contrastingly, for PC and PS deficiencies, the MDR dropped significantly and mildly lowered to subnormal values. At PS activities >55% for PS no mutations were detected. Mutation profiles of all three genes were similar with the highest prevalence for missense mutations (63-78%), followed by nonsense (7-11%), splice-site mutations (7-13%), small deletions (1-8%), small insertions/duplications (1-4%) and large deletions (3-6%). In conclusion, genetic testing is a useful diagnostic tool for diagnosing thrombophilia. Based on our data, genetic analysis for patients with AT deficiency is indicated for all subnormal activities. In contrast, genotyping is not advisable for PC activities >70% and for PS activities >55%.
Thrombosis and Haemostasis 05/2012; 108(2):247-57. · 5.04 Impact Factor
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Ingrid Bartsch,
Kirstin Sandrock,
Francois Lanza,
Paquita Nurden,
Ina Hainmann, Anna Pavlova,
Andreas Greinacher,
Uta Tacke,
Michael Barth,
Anja Busse,
Johannes Oldenburg,
Martin Bommer,
Brigitte Strahm,
Andrea Superti-Furga,
Barbara Zieger
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ABSTRACT: The bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5' to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient's platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealed impaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleeding episodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.
Thrombosis and Haemostasis 07/2011; 106(3):475-83. · 5.04 Impact Factor
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Haemophilia 07/2011; 18(1):e3-4. · 2.60 Impact Factor
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Journal of Thrombosis and Haemostasis 03/2011; 9(5):1083-6. · 5.73 Impact Factor
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Beate Luxembourg,
Daniel Delev,
Christof Geisen,
Michael Spannagl,
Manuela Krause,
Wolfgang Miesbach,
Christine Heller,
Frauke Bergmann,
Ursula Schmeink,
Ralf Grossmann,
Edelgard Lindhoff-Last,
Erhard Seifried,
Johannes Oldenburg, Anna Pavlova
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ABSTRACT: Antithrombin (AT) is the most important physiological inhibitor of coagulation proteases. It is activated by glycosaminoglycans such as heparin. Hereditary antithrombin deficiency is a rare disease that is mainly associated with venous thromboembolism. So far, more than 200 different mutations in the antithrombin gene (SERPINC1) have been described. The aim of our study was to characterise the molecular background in a large cohort of patients with AT deficiency. Mutation analysis was performed by direct sequencing of SERPINC1 in 272 AT-deficient patients. Large deletions were identified by multiplex PCR coupled with liquid chromatography or multiplex ligation-dependent probe amplification (MLPA) analysis. To predict the effect of SERPINC1 sequence variations on the pathogenesis of AT deficiency, in silico assessments, multiple sequence alignment, and molecular graphic imaging were performed. The mutation profile consisted of 59% missense, 10% nonsense, 8% splice site mutations, 15% small deletions/insertions/duplications, and 8% large deletions. Altogether 87 different mutations, including 42 novel mutations (22 missense and 20 null mutations), were identified. Of the novel missense mutations, nine are suspected to impair the conformational changes that are needed for AT activation, two to affect the central reactive loop or the heparin binding site, and six to impair the structural integrity of the molecule. Despite the heterogeneous background of AT deficiency, 10 AT variants occurred in multiple index patients. Characterisation of the SERPINC1 mutation profile in large cohorts of patients may help to further elucidate the pathogenesis of AT deficiency and to establish genotype-phenotype associations.
Thrombosis and Haemostasis 01/2011; 105(4):635-46. · 5.04 Impact Factor
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Hamostaseologie 11/2010; 30(41):S153-5. · 1.19 Impact Factor
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ABSTRACT: Severity of bleeding phenotype in hemophilia A (HA) depends on the underlying mutation in the F8 gene and, ultimately, on the concentration and functional integrity of the factor VIII (FVIII) protein in circulating plasma. Initial diagnosis for HA and monitoring of treatment is typically performed by measuring of FVIII activity by either one-stage assay or chromogenic assay. We review evidence for why both types of assay do not give comparable results in a significant proportion of patients with non-severe haemophilia A and why the discrepancy in results between both methods segregates with distinct subclasses of known missense mutations causing haemophilia A. The current understanding of the mechanistic basis for how FVIII:C assay discrepancies arise are discussed. Conclusion: We propose that both methods should be used in initial patient diagnosis along with follow-up genetic analysis to avoid potential misdiagnosis and to optimize treatment monitoring of patients with HA phenotypes.
Hamostaseologie 11/2010; 30(4):207-11. · 1.19 Impact Factor
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L Salazar-Sánchez,
G Jiménez-Cruz,
M Mendez,
P Chaverri,
P Alvarado,
W Schröder,
K Wulff,
M Sandoval,
F H Herrmann, A Pavlova,
J Oldenburg
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ABSTRACT: Haemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII (FVIII). It is caused by FVIII gene intron 22 inversion (Inv22) in approximately 45% and by intron 1 inversion (Inv1) in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The aim of this study was to analyze the presence of these mutations in 25 HA Costa Rican families. Patients, methods: We studied 34 HA patients and 110 unrelated obligate members and possible carriers for the presence of Inv22or Inv1. Standard analyses of the factor VIII gene were used incl. Southern blot and long-range polymerase chain reaction for inversion analysis. Results: We found altered Inv22 restriction profiles in 21 patients and 37 carriers. It was found type 1 and type 2 of the inversion of Inv22. During the screening for Inv1 among the HA patient, who were Inv22 negative, we did not found this mutation. Discussion: Our data highlight the importance of the analysis of Inv22 for their association with development of inhibitors in the HA patients and we are continuous searching of Inv1 mutation. This knowledge represents a step for genetic counseling and prevention of the inhibitor development.
Hamostaseologie 11/2010; 30 Suppl 1:S150-2. · 1.19 Impact Factor
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ABSTRACT: The genotype-phenotype relationship of compound heterozygous protein S-deficiency in a 7-year-old girl with reduced protein S-levels and a severe cerebral sinovenous thrombosis is illustrated. In this patient we identified a novel deletion in the protein S-gene causing a compound heterozygous state and subsequently a symptomatic protein S-deficiency. In case of thrombosis analysis of protein S is recommended. Low levels of protein S should be further investigated by molecular diagnostics.
Klinische Pädiatrie 05/2010; 222(3):194-5. · 1.77 Impact Factor
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ABSTRACT: Acquired haemophilia A (AH) is a rare bleeding disorder caused by an auto-antibody to coagulation factor VIII. It is associated with various autoimmune diseases, pregnancy, cancer or drug ingestion; however, in 50% of patients, no underlying disorder is found. In the present study, we investigated the association of HLA class I (A, B and Cw) and class II (DRB1 and DQB1) alleles with AH in a cohort of 57 patients. While no association with any class I allele was detected, a significantly higher frequency of DRB1*16 [odds ratio (OR) 10.2, 95%CI: 5.32-19.57, P < 0.0001] and DQB1*0502 (OR 2.2, 95%CI: 1.12-4.54, P < 0.05) was observed. In contrast, the frequency of DRB1*15 and DQB1*0602 alleles was found to be decreased in patients with AH corresponding to an OR of 0.4 for both HLA loci. Upon comparing the frequencies of these alleles with those of patients with congenital haemophilia A with inhibitors, the data demonstrate that the high risk alleles in patients with AH DRB1*16 and DQB1*0502 are found to be low risk alleles in patients with congenital haemophilia A with inhibitors (OR 1.1 and 1.5 respectively). Conversely, the alleles that exhibit low risk in AH DRB1*15 and DQB1*0602 are found to be high risk for haemophilia A inhibitor patients (OR 2.2 and 3.7 respectively). The pathophysiological reason for this finding remains unknown. It might be speculated that the presence or absence of the FVIII antigen and the various ability of HLA molecules to present the FVIII antigen to the T-cell receptor contribute to these findings.
Haemophilia 05/2010; 16(102):107-12. · 2.60 Impact Factor
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ABSTRACT: Acquired haemophilia (AH) is a rare autoimmune bleeding disorder, which arises as a result of the spontaneous production of autoantibodies against endogenous factor VIII. The breakdown in immune tolerance is thought to be a result of a combination of genetic and environmental factors. Both human leucocyte antigen (HLA) and cytotoxic T lymphocyte antigen 4 (CTLA-4) play an important role in the maintenance of peripheral T-cell tolerance. A higher frequency of HLA class II alleles and single nucleotide polymorphisms of the CTLA-4 gene have been observed in some autoimmune diseases and severe haemophilia A. In 57 patients with AH, significantly higher frequencies of the HLA class II alleles DRB*16 [odds ratio (OR) 10.2] and DQB1*0502 (OR 2.5) have been detected when compared with controls. The CTLA-4 + 49 G allele has also presented with a significantly higher frequency in the same cohort of patients with AH (OR 2.17). This observation was mainly because of a higher frequency of the CTLA-4 + 49 G allele in female patients. These findings suggest that immune response genes may contribute to the development of anti-factor VIII autoantibodies in AH.
Haemophilia 05/2010; 16 Suppl 3:41-5. · 2.60 Impact Factor
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ABSTRACT: Haemophilia A (HA) is a common, X-linked, recessive disorder caused by defi- ciency or dysfunction of coagulant factor VIII. The gene encoding this factor is large and complex (186 kb and 26 exons). It is situated at about 1 Mb from the telomere at the Xq28 (1). Various types of mutations in the factor VIII gene are responsible for the bleeding disorder.
Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change is found, even after sequencing the whole coding part of the F8 gene including the flanking splice sites, as well as the promoter and the 3' UTR regions (2, 3). Therefore, mutations or rearrangements in non-coding areas or even in other loci have to be considered re- sponsible for the haemophilia A pheno- type. In this study we analyzed 15 such pa- tients.Weusedalong-rangePCRscreening method to cover 190 kb that include the whole genomic region of F8.
Hamostaseologie 01/2010; 30(4a):S158-S161. · 1.19 Impact Factor
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ABSTRACT: Background: Approximately 25% of severe hemophilia A (HA) patients develop antibodies to factor VIII protein. Patients: In the present case-controlled cohort study, 260 severely affected, mutation-type-matched HA patients were studied for association of human leukocyte antigen (HLA) class II molecules and polymorphisms in the genes encoding interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) and development of inhibitors. Results: Our results demonstrate a higher frequency of DRB1*15 and DQB1*0602 alleles as well as of the haplotype DRB1*15/DQB1*0602 in inhibitor patients [odds ratio (OR) 1.9; P < 0.05]. In TNF-α, the A allele of the −308G>A polymorphism was found with higher frequency in the inhibitor cohort (0.22 vs. 0.13, OR 1.80). This finding was more pronounced for the homozygous A/A genotype (OR 4.7). For IL-10, the −1082G allele was observed more frequently in patients with inhibitors (0.55 vs. 0.43; P = 0.008). The functional cytokine phenotype was determined for the first time, on the basis of the genetic background, and this showed that 12% of patients with inhibitors were high-TNF-α/high-IL-10 producers, as compared with 3% of non-inhibitor patients (OR 4.4). A trend for a lower frequency of the A allele of the CT60 polymorphism in CTLA-4 was found in inhibitor patients (0.42 vs. 0.50). Conclusions: In conclusion, the reported data clearly highlighted the participation of HLA molecules in inhibitor formation in a large cohort of patients. The higher frequencies of the −308G>A polymorphism in TNF-α and −1082A>G in IL-10 in inhibitor patients confirmed the earlier published data. The CT60 single-nucleotide polymorphism in CTLA-4 is of apparently less importance.
Journal of Thrombosis and Haemostasis 10/2009; 7(12):2006 - 2015. · 5.73 Impact Factor
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ABSTRACT: . Background: Approximately 25% of severe hemophilia A (HA) patients develop antibodies to factor VIII protein. Patients: In the present case-controlled cohort study, 260 severely affected, mutation-type-matched HA patients were studied for association of human leukocyte antigen (HLA) class II molecules and polymorphisms in the genes encoding interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) and development of inhibitors. Results: Our results demonstrate a higher frequency of DRB1*15 and DQB1*0602 alleles as well as of the haplotype DRB1*15/DQB1*0602 in inhibitor patients [odds ratio (OR) 1.9; P < 0.05]. In TNF-alp[ha, the A allele of the 308G>A polymorphism was found with higher frequency in the inhibitor cohort (0.22 vs. 0.13, OR 1.80). This finding was more pronounced for the homozygous A/A genotype (OR 4.7). For IL-10, the 1082G allele was observed more frequently in patients with inhibitors (0.55 vs. 0.43; P = 0.008). The functional cytokine phenotype was determined for the first time, on the basis of the genetic background, and this showed that 12% of patients with inhibitors were high-TNF-alpha/high-IL-10 producers, as compared with 3% of non-inhibitor patients (OR 4.4). A trend for a lower frequency of the A allele of the CT60 polymorphism in CTLA-4 was found in inhibitor patients (0.42 vs. 0.50). Conclusions: In conclusion, the reported data clearly highlighted the participation of HLA molecules in inhibitor formation in a large cohort of patients. The higher frequencies of the 308G>A polymorphism in TNF-alpha and 1082A>G in IL-10 in inhibitor patients confirmed the earlier published data. The CT60 single-nucleotide polymorphism in CTLA-4 is of apparently less importance.
Journal of Thrombosis and Haemostasis 10/2009; 7(12):2006-2015. · 5.73 Impact Factor
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ABSTRACT: Coagulation factor V (FV) plays an important role in the blood coagulation cascade as part of the prothrombinase complex. FV deficiency is a rare autosomal recessive bleeding disorder with variable phenotypic expression. Thus, our study reports 39 patients with FV deficiency. In 36 cases, we were able to identify a causative mutation. Of these, 20 patients were heterozygous for the identified mutation, nine were homozygous, six were compound heterozygous and one proband was pseudohomozygous. In the remaining patients, no mutation was found. A total of 42 genetic alterations (of which 33 were uniquely different mutations), comprising 19 missense mutations, eight nonsense mutations, four small deletions and two splice site mutations, were identified by this study. Twenty-three of these were novel sequence variations not previously described in the literature. Interestingly, all changes found in exon 13 resulted in null alleles as either nonsense mutations or small deletions. The overall profile of these new mutations corresponds well with the data published in the F5 database. In those cases, where data were available, information on FV activity levels and/or bleeding history is given. Interestingly, some patients with mild FV deficiency (FV:C about 50% of normal) also exhibited bleeding episodes. Our data substantially contribute to the broadening and better understanding of the FV deficiency mutational spectrum. Identifying the molecular basis of mutations underlying this rare coagulation disorder will allow more insight into the mechanisms involved in the variable clinical phenotypes of patients with FV deficiency.
Haemophilia 06/2009; 15(5):1143-53. · 2.60 Impact Factor
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ABSTRACT: The genotype-phenotype relationship of compound heterozygous factor X deficiency in a young girl with severe factor X deficiency and bleeding symptoms is characterized. We identified a novel deletion of exon 6 and a missense mutation (c.856G>A, Val286Met) in exon 7 of the F10 gene leading to a compound heterozygous state and causing severe factor X deficiency. Therapeutic options for patients with symptomatic factor X deficiency are demonstrated.
Hamostaseologie 05/2009; 29(2):184-6. · 1.19 Impact Factor
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ABSTRACT: Hemophilia A (HA) in females is a rare observation. Here we describe various genetic mechanisms that result in phenotypic expression of HA in seven females.
The F8 gene was examined in all patients and relatives by direct sequencing. Multiplex ligation-dependent probe amplification (MLPA) was performed for large deletion screening. X chromosome inactivation was studied by PCR analysis of a polymorphic CAG repeat in the first exon of the human androgen receptor (HUMARA) gene.
In two females sequencing of the F8 gene revealed homozygous missense mutations (Arg593Cys and Tyr1680Phe) as a consequence of consanguineous marriage. The third case was due to compound heterozygosity comprising the missense mutation Leu412Phe inherited from the carrier mother, together with a de novo large deletion spanning exon 9-22, probably originating from the germ cells of the healthy father. Three further cases shared a common mechanism representing heterozygous mutations in the F8 gene (Arg1781His, Arg327His, small deletion in exon 10) combined with non-random inactivation of the X chromosome. The final case describes a coincidental inheritance of HA and Coffin-Lowry syndrome in the same family. The HA phenotype results from a heterozygous small deletion affecting the F8 gene (c.6872 del CT leading to Thr2272fs) and a complete inactivation of the maternal X chromosome, which segregates with Coffin-Lowry syndrome in the two brothers of the proposita.
In conclusion, molecular genetic analysis represents an essentially valuable tool in elucidating the nature of the molecular mechanisms underlying the HA phenotype in females.
Journal of Thrombosis and Haemostasis 04/2009; 7(6):976-82. · 5.73 Impact Factor
