Seven novel and four recurrent point mutations in the factor VIII (F8C) gene

Haemophilia A is a X‐linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are “private”, because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype‐phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated. © 2001 Wiley‐Liss, Inc.     

The identification and classification of 41 novel mutations in the factor VIII gene (F8C)

Hemophilia A is a bleeding disorder caused by a quantitative or qualitative deficiency in the coagulation factor VIII. Causative mutations are heterogeneous in nature and are distributed throughout the FVIII gene. With the exception of mutations that result in prematurely truncated protein, it has proved difficult to correlate mutation type/amino acid substitution with severity of disease. We have identified 81 mutations in 96 unrelated patients, all of whom have typed negative for the common IVS-22 inversion mutation. Forty-one of these mutations are not recorded on F8C gene mutation databases. We have analyzed these 41 mutations with regard to location, whether or not each is a cross-species conserved region, and type of substitution and correlated this information with the clinical severity of the disease. Our findings support the view that the phenotypic result of a mutation in the FVIII gene correlates more with the position of the amino acid change within the 3D structure of the protein than with the actual nature of the alteration.     

High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype-phenotype correlation

Hemophilia A (HEMA) is an X-linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular genetic testing for the factor VIII gene is challenging due to its large size. Here we present results of high throughput mutation scanning based on Southern blot analysis and direct sequencing of all PCR amplified coding exons and the exon-intron boundaries of the factor VIII gene. The results of mutation analysis on 89 hemophiliac males showed presence of a disease-causing mutation in 80 individuals (90%, 95% CI of 82%-95%). Seven out of nine mutation-negative individuals were severe cases of hemophilia A with < 1% factor VIII protein in the blood. The correlation of phenotype with genotype as observed in this study was not absolute. This finding is supported by similar observations in the international database for hemophilia A mutations (HAMSTeRS). This issue raises the importance of genotypes at other loci that can act as modifiers for the phenotype. Thirty-four novel mutations and three novel substitutions for previously reported amino acid residues were identified in this series of 80 mutations. The mutations cover the full spectrum including rearrangements, deletions, frameshift, and point mutations. The novel missense mutations require careful evaluation. Prediction of a mutation as the disease-causing allele was made from the nature of the substitution and the degree of conservation of the mutated amino acid among species that have diverged in evolution. In some cases segregation analysis of the mutation with disease condition was performed when other family members were available.     

Detection of 95 novel mutations in coagulation factor VIII gene F8 responsible for hemophilia A: results from a single institution

Hemophilia A (HA) is an X‐linked hereditary bleeding disorder defined by a qualitative and/or quantitative factor VIII (FVIII) deficiency. The molecular diagnosis of HA is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. The putative role of the novel mutations, especially missense mutations, may be difficult to interpret as causing HA. We identified 95 novel mutations out of 180 different mutations responsible for HA in 515 patients from 406 unrelated families followed up at a single hemophilia treatment center of the Bicêtre university hospital (Assistance Publique‐Hôpitaux de Paris [AP‐HP], Le Kremlin‐Bicêtre). These 95 novel mutations comprised 55 missense mutations, 12 nonsense mutations, 11 splice site mutations, and 17 small insertions/deletions. We therefore developed a mutation analysis based on a body of proof that combines the familial segregation of the mutation, the resulting biological and clinical HA phenotype, and the molecular consequences of the amino acid (AA) substitution. For the latter, we studied the putative biochemical modifications: its conservation status with cross‐species FVIII and homologous proteins, its putative location in known FVIII functional regions, and its spatial position in the available FVIII 3D structures. The usefulness of such a strategy in interpreting the causality of novel F8 mutations is emphasized. Hum Mutat 27(7), 676–685, 2006. © 2006 Wiley‐Liss, Inc.     

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

Hemophilia A is a common X-linked bleeding disorder caused by various types of mutations in the factor VIII gene F8C. The most common intron 22-inversion is responsible for about 40% of the severe hemophilia A cases while large deletions, point mutations and small (less than 100 bp) deletions or insertions are responsible for the disease in the rest of patients. We report on nine novel (6 deletions, two indels and one partial duplication) and five recurrent small rearrangements identified in 15 German patients with severe hemophilia A, negative for the intron 22-inversion. c.2208-2214delTTATTAC/c.2207-2215insCTCTT and c.4665-4678del/c.4664-4678insAAGGAA identified in the present study are the first small indels described in the factor VIII gene. Our analyses suggest that the prevalence of this type of mutations (predominantly located in exon 14) among patients with severe phenotype and negative for the common intron 22-inversion, is about 30%. The correlation between these molecular defects and formation of factor VIII inhibitors as well as the parental origin of the de novo mutations are evaluated. Finally we show that denaturing HPLC (DHPLC) and classic heteroduplex analysis (HA) are able to detect these sequence alterations on 100% and could be preferred as a screening approach when analysing for mutations in factor VIII in severely affected patients.     

Mutational‐screening in the factor VIII gene resulting in the identification of three novel mutations,one of which is a donor splice mutation

Hemophilia A is an X‐linked bleeding disease caused by mutations in the coagulation factor VIII gene. The identification and characterization of pathogenic mutations allows the recognition of new mechanisms of functional disturbances of factor VIII. To screen for mutations exons 1‐26 of the factor VIII gene have been amplified genomically and analyzed by SSCP followed by direct sequencing of respective exons showing abnormal electrophoretic mobility on SSCP analysis. In the present study we report the detection of four mutations in the factor VIII gene, of which three are novel. The mutational analysis of a patient with severe hemophilia A has revealed that the a ®c transversion at position 3 of the donor‐splice‐site of intron 23 results in the skipping of exon 23. A novel nonsense mutation Q1778X in exon 16 of factor VIII gene has been identified in a second hemophilia A case. Furthermore two missense mutations have been ascertained: a novel, S183R, causing a mild phenotype of hemophilia A and R282H, previously described in association with severe hemophilia A. Hum Mutat 13:504, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of seven novel mutations of F8C by DHPLC

Hemophilia A is an X-linked recessive disorder resulting from deficiency of Factor VIII (F8C), an important protein in blood coagulation. A large number of disease producing mutations have been reported in the F8C gene. However, a comprehensive analysis of mutations is difficult to conduct due to the large gene size, its many scattered exons, and the high frequency of de novo mutations. In this study, we performed analysis using PCR, Conformation Sensitive Gel Electrophoresis (CSGE), Denaturing High Performance Liquid Chromatography (DHPLC) and direct sequencing. We found seven novel mutations causing severe, moderate and mild Hemophilia A: IVS14-1G>A, G458V, T1695S, L1758P, Q2311P, 1441delT, 1269-1271insA. At least four variants detected by DHPLC (IVS14-1G>A, Q2311P,_R698W and D1241Q) were not detectable by CSGE.     

Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication. Hum Mutat 12:393–402, 1998. © 1998 Wiley-Liss, Inc.     

Maternally transferred anti-factor VIII IgG reduce the anti-factor VIII humoral immune response in factor VIII-deficient mice

Replacement therapy with exogenous factor VIII (FVIII) to treat haemorrhages or used in prophylaxis induces inhibitory anti‐FVIII immunoglobulin G (IgG) in some patients with haemophilia A. Therapeutic strategies to prevent the onset of the deleterious anti‐FVIII immune response are still lacking. Maternal IgG is transferred to the offspring during fetal and neonatal life. While protecting the offspring from bacterial and viral infections, maternal IgG may alter the repertoires of T and B lymphocytes, and may impair vaccination in early infancy. Using haemophilic mice, we demonstrate that the transfer of maternal anti‐FVIII IgG modulates the onset of anti‐FVIII inhibitory IgG in early adulthood. The protective effect is reproduced upon reconstitution of naive mice with anti‐FVIII IgG, suggesting that the reduced ability to mount an anti‐FVIII immune response is the result of an interference between circulating anti‐FVIII IgG and the administered FVIII rather than to a profound remodelling of lymphocyte repertoires occurring during the ontogeny of the immune system.     

F8基因倒位检测及其在甲型血友病基因诊断中的应用

目的建立F8基因第22内含子倒位突变检测新方法,应用于甲型血友病(hemophilia A)基因诊断。方法应用长距离PCR(long distance-polymerase chain reaction,LD-PCR)、倒位PCR(inversion-PCR,IPCR)技术检测31例甲型血友病患者F8基因22内含子倒位;对于倒位突变阳性患者的母亲应用上述两种方法进行携带者诊断;而对倒位携带者孕妇于孕中期抽取羊水,进行产前基因诊断。结果31例甲型血友病患者中查出7例存在倒位突变;4例倒位突变阳性患者的母亲有3例为倒位携带者;对1例倒位携带者孕妇进行了产前诊断,确定其胎儿无倒位突变。结论LD-PCR、I-PCR技术可快速检测F8基因22内含子倒位突变,可应用于患者及携带者基因诊断;I-PCR可用于F8基因22内含子倒位的产前基因诊断。     

Phenotypical spectrum of cerebellar ataxia associated with a novel mutation in the CA8 gene,encoding carbonic anhydrase (CA) VIII

We define the neurological characteristics of familial cases from multiple branches of a large consanguineous family with cerebellar ataxia, mental retardation (MR), and dysequilibrium syndrome type 3 caused by a mutation in the recently cloned CA8 gene. The linkage analysis revealed a high logarithm of the odds (LOD) score region on 8q that harbors the CA8 in which a novel homozygous c.484G>A (p.G162R) mutation was identified in all seven affected members. The patients had variable cerebellar ataxia and mild cognitive impairment without quadrupedal gait. The brain MRI showed variable cerebellar volume loss and ill‐defined peritrigonal white matter abnormalities. The Fluorodeoxyglucose Positron Emission Tomography (FDG PET) revealed hypometabolic cerebellar hemispheres, temporal lobes, and mesial cortex. This report expands the neurological and radiological phenotype associated with CA8 mutations. CA8 involvement should be considered in the differential diagnosis of other genetically unresolved autosomal recessive cerebellar ataxias. © 2011 Wiley‐Liss, Inc.     

First trimester prenatal diagnosis of haemophilia a using factor VIII gene probe

Accurate first-trimester prenatal diagnosis was achieved in a Japanese haemophilia A family by the use of a restriction fragment length polymorphism (RFLP) located within the F.VIII gene. Since the pregnant woman's heterozygosity forBclI polymorphism in F.VIII/intron 18 (F8A) probe was informative, chorionic villus sampling (CVS) was performed at 9 weeks of gestation. Restriction analysis showed that the fetus was heterozygous for theBclI site and had received a normal paternal X chromosome (0.9 kb) and a normal maternal X (1.2 kb). Therefore, we concluded that the fetus was a non-carrier female. Pregnancy went to term and woman gave birth to an apparently healthy female. At one week after birth a coagulation study confirmed that the newborn infant is not a carrier. The first-trimester prenatal diagnosis of haemophilia A is possible by CVS due to a RFLP in the F.VIII gene.     

Molecular diagnostics of 15 hemophilia A patients: Characterization of eight novel mutations in the factor VIII gene,two of which result in exon skipping

The X-linked bleeding disorder hemophilia A is caused by mutations in the coagulation factor VIII gene. A high frequency of de novo mutations and the large size of this gene complicate the molecular diagnostic of hemophilia A. Characterization of mutations, however, may help identify amino acids or regions with essential functional or structural properties and thereby clarify the mechanism of pathogenesis. In the present study, we describe the identification of 15 mutations in the factor VIII gene, of which eight are novel. Among the patients with severe hemophilia A, two splice mutations (IVS5-3 and IVS19-2), a 4-bp deletion (TACA) at codon 1215, and a missense mutation G1850V have been characterized. The missense mutations G479R, R531C, V537D, N2129S and I2190N were found for five patients with a moderate course of hemophilia A disease. A silent mutation resulting in activation of a cryptic acceptor splice site within exon 11 and four other missense mutations Y114C, R1689H, R2150H (2x), M2164V have been identified for six patients with mild hemophilia A. Hum Mutat 12:301–303, 1998.© 1998 Wiley-Liss, Inc.     

Three cases of acquired factor VIII: C inhibitors in non-hemophilic patients

Summary During the last ten years we observed three non-hemophilic patients with factor(F) VIII: C inhibitors (2 women aged 68 and 80 and a man aged 51).In all three cases, a sudden bleeding tendency was observed shortly after an injury or surgery. Coagulation tests showed a prolonged aPTT and a decreased F VIII: C level. Other deficiencies of blood-clotting factors and acquired or hereditary von Willebrand's disease could be excluded. Therapy with F VIII: C concentrate, cryoprecipitate, or fresh-frozen plasma did not produce the expected increase in F VIII: C. Measurement of F VIII: C inhibitor levels (Bethesda Units, BU) revealed values in the range between 9 and 64 BU. The two patients subjected to long-term therapy with a combination of prednisone (initially 2–3 mg/kg BW) and azathioprine (2–3 mg/kg BW) responded positively; the F VIII: C concentration increased. The third patient, treated only with a low dose of prednisone (30 mg/day), did not show any reaction at all. Since hereditary hemophilia A could be excluded, the inhibitors apparently were acquired. Malignant tumors did not appear. In conclusion, long-term therapy of an acquired F VIII: C inhibitor with a combination of prednisone and azathioprine may lead to complete disappearance of the inhibitor, normalization of the coagulation tests, and complete remission of the bleeding tendency.Abbreviations aPCC activated prothrombin complex concentrate - aPTT activated partial thromboplastin time - BU Bethesda Units - BW body weight - CP cryoprecipitate - DIC disseminated intravascular coagulation - F III: C factor VIII procoagulant activity - FEIBA factor eight inhibitor bypassing activity - FFP fresh-frozen plasma     

Histocompatibility antigens and alleles in Japanese haemophilia A patients with or without factor VIII antibodies

We carried out human leukocyte antigen (HLA)-A, B, Cw, DR and DQ serological typing and HLA-DQA1, DQB1, DRB1 and DPB1 genetic typing for 46 Japanese haemophilia A patients, including 20 who had developed an antibody to factor VIII. It appears that anti FVIII inhibitor formation is associated with the major histocompatibility complex in Japanese haemophilia A patients. Absence of HLA-A24 is a principal risk factor for inhibitor formation in Japanese haemophilia A patients. As supplemental risk factors, HLA-DR4.1, DQ4 and DQA1*0301=2 are positively associated with patients exhibiting inhibitor compared with normal subjects. This and previous studies show that the association between HLA antigens and the formation of inhibitor depends on race. Data of HLA typing may be useful for the recognition of groups at high risk for the possible formation of inhibitor among Japanese haemophilia A patients.     

Mutations in the factor IX gene (F9) during the past 150 years have relative rates similar to ancient mutations

Pollutants and dietary mutagens have been associated with somatic mutation and cancer, but the extent of their influence on germline mutation is not clear. Since deleterious germline mutations can be transmitted for thousands of years, any influence on germline mutation from the vast increase in man‐made chemicals of the past 150 years would be an important public health issue. Observed disease causing mutations in the X‐linked factor IX gene (F9) of hemophilia B patients originated predominantly in the past 150 years, since the half‐life of these mutations in human populations had been about two generations before effective treatment became available about a generation ago. Recent changes in germline mutational processes may be detected by comparison of the observed hemophilia B causing mutation pattern in F9 with the pattern of neutral polymorphisms which occurred over a much longer period of time. By scanning a total of 1.5 megabases of deep intronic regions of F9 in the genomic DNA from 84 individuals, 42 neutral polymorphisms were found in 23 haplotypes that differed by at least 11 mutations from the ancestral primate haplotype. By sequencing F9 in seven non‐human primates, 39 of these polymorphisms were characterized as ancient mutations relative to a unanimous ancestral primate allele. This ancient mutation pattern was compared to the recent pattern of hemophilia B causing mutations. Remarkably, no significant difference was found (P=0.5), suggesting that the vast increase in man‐made chemicals during the past 150 years has not had a major impact on the pattern of human germline mutation. This result is consistent with the hypothesis that endogenous processes dominate germline mutation. Hum Mutat 19:49–57, 2002. © 2001 Wiley‐Liss, Inc.     

Seven novel point mutations in the uroporphyrinogen decarboxylase (UROD) gene in patients with familial porphyria cutanea tarda (f-PCT)

In this work, we describe seven novel molecular defects in the uroporphyrinogen decarboxylase gene responsible for familial porphyria cutanea tarda in Italian subjects with reduced erythrocyte URO-D activity. Four of these molecular abnormalities (R142Q, L161Q, S219F, P235S) are missense mutations, one (Q206X) is a nonsense mutation, one (IVS8-1 G>C) is a splicing defect causing the exon 9 deletion and one (1107 G>A) is located in the 3' untranslated region of UROD gene. All the amino acid substitutions fall in conserved regions in several organisms suggesting an important role in catalysis or in the protein structure stabilization. Three of these mutations have been detected in more than one subject. These results suggest a molecular heterogeneity at the UROD locus in Italian PCT patients although recurrent mutations have been identified.     

Common intragenic and extragenic polymorphisms of blood coagulation factors VIII and IX are different in Chinese and Caucasian populations

In order to examine the possibilities of carrier detection and prenatal diagnosis in hemophilia A and B in the Chinese region of Suzhou, we analyzed four different RFLPs within the factor IX gene and two intragenic RFLPs and one extragenic RFLP for the factor VIII gene. The results obtained show important differences between the Chinese and Caucasian populations. No polymorphism was found within the factor IX gene in the Chinese population and the informativity obtained for the factor VIII gene was quite different between the two populations for each RFLP studied.