Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients

The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.     

New Insight into the Molecular Basis of Hemophilia A

Since publication of the sequence of the factor VIII gene (F8) in 1984, a large number of mutations that cause hemophilia A (HA) have been identified. With the technical advances associated with mutation screenings, it is now possible to identify a putative F8 sequence alteration in the great majority of HA patients. The mutation spectrum includes 2 inversion hot spots (intron 1 and intron 22 inversions) mediated by intrachromosomal recombination between 2 copies of long inverted repeats, one of which lies within the F8 gene whereas the other is extragenic. Point mutations are distributed over all of the exons, and deletions or insertions of different sizes and mutations affecting splice sites account for the rest of the known mutations. In a small number of cases, however, we are unable to find any disease-determining DNA changes in the coding regions of the F8 gene. This fact points to possibilities of unknown gene rearrangements that disrupt the F8 gene or mutations in other genes that play a role in the processing/secretion of the factor VIII protein. Moreover, the proof of an absence of F8 messenger RNA (mRNA) in one patient points to either a defect in the expression of F8 mRNA or its rapid degradation, which may represent a novel mechanism leading to HA.     

Direct detection of a common inversion mutation in the genetic diagnosis of severe hemophilia A [see comments]

Two recent reports suggest that approximately 50% of the cases of severe hemophilia A (factor VIII:C < 0.01 U/mL) may be caused by a gross rearrangement of the factor VIII gene. The mutation involves genomic sequence from exon 1 to within intron 22 of the gene in an inversion event. This rearrangement can be detected on a Southern blot using a probe that is complementary to sequence from within intron 22. In this report, we describe the analysis of 71 severe hemophilia A patients for the presence of this mutation. Thirty-two of the patients (45%) showed evidence of the rearrangement, a figure that confirms the initial reports on 28 patients. Five different patterns of rearrangement have been noted, although two of these patterns (pattern 1 [70%] and pattern 2 [16%]) account for the majority of cases. The other patterns of rearrangement appear to be confined to individual families and may represent the result of additional sequence variation within the region of the genome to which the proximal 22 exons of factor VIII are translocated. Analysis of this patient population for the factor VIII inversion mutation has been extremely useful in a molecular diagnostic sense. In 23 of the cases studied (72%), the affected individual was the only documented hemophiliac in the family and, thus, previous linkage analysis had been limited to the provision of exclusion testing only. In conclusion, it appears that testing for the factor VIII inversion mutation will be positive in approximately 45% of severe hemophiliacs and as such should constitute the initial stage in the genetic testing protocol for these patients' families.     

A deletion mutation causes hemophilia B in Lhasa Apso dogs

Hemophilia B is a bleeding disorder caused by a deficiency of clotting factor IX (FIX). A colony of FIX deficient Lhasa Apso dogs has been established and the molecular basis of hemophilia B has been determined. The plasma factor IX levels were < 1% of normal canine levels in affected dogs. A complex deletion mutation at nucleotides 772- 777 was found when hepatocyte cDNA from a hemophilia B dog was sequenced. The sequence was identical to the normal canine sequence except for a deletion including nucleotides 772-776 and a C-->T transition at nucleotide 777. The mutation results in mRNA instability and a premature termination codon in the nucleotide sequence encoding the activation peptide. The mutation was verified by sequencing genomic DNA from an FIX-deficient dog. A genetic test for the detection of heterozygous animals was established using heteroduplex analysis. Although hemophilia B has been described in many dog breeds, this is only the second mutation to be sequenced. The Lhasa Apso dog model should be valuable for evaluating novel strategies for treating hemophilia B such as gene therapy.     

Factor 8 (F8) gene mutation profile of Turkish hemophilia A patients with inhibitors.

Factor VIII (FVIII) replacement therapy is ineffective in hemophilia A patients who develop alloantibodies (inhibitors) against FVIII. The type of factor 8 (F8) gene mutation, genes in the major histocompatibility complex loci, and also polymorphisms in IL-10 and tumor necrosis factor-alpha are the major predisposing factors for inhibitor formation. The present study was initiated to reveal the F8 gene mutation profile of 30 severely affected high-responder patients with inhibitor levels of more than 5 Bethesda U (BU)/ml and four low-responder patients with inhibitors less than 5 BU/ml. Southern blot and PCR analysis were performed to detect intron 22 and intron 1 inversions, respectively. Point mutations were screened by DNA sequence analysis of all coding regions, intron/exon boundaries, promoter and 3' UTR regions of the F8 gene. The prevalent mutation was the intron 22 inversion among the high-responder patients followed by large deletions, small deletions, and nonsense mutations. Only one missense and one splicing error mutation was seen. Among the low-responder patients, three single nucleotide deletions and one intron 22 inversion were found. All mutation types detected were in agreement with the severe hemophilia A phenotype, most likely leading to a deficiency of and predisposition to the development of alloantibodies against FVIII. It is seen that Turkish hemophilia A patients with major molecular defects have a higher possibility of developing inhibitors.     

Unbalanced X-chromosome inactivation with a novel FVIII gene mutation resulting in severe hemophilia A in a female

This report is of a 14-month-old girl affected with severe hemophilia A. Both her parents had normal values for factor VIII activity, and von Willebrand disease type 2N was excluded. Karyotype analysis demonstrated no obvious alteration, and BclI Southern blot did not reveal F8 gene inversions. Direct sequencing of F8 gene exons revealed a frameshift-stop mutation (Q565delC/ter566) in the heterozygous state in the proposita only. F8 gene polymorphism analysis indicated that the mutation must have occurred de novo in the paternal germline. Furthermore, analysis of the pattern of X chromosome methylation at the human androgen receptor gene locus demonstrated a skewed inactivation of the derived maternal X chromosome from the lymphocytes of the proband's DNA. Thus, the severe hemophilia A in the proposita results from a de novo F8 gene frameshift-stop mutation on the paternally derived X chromosome, associated with a nonrandom pattern of inactivation of the maternally derived X chromosome. (Blood. 2000;96:4373-4375)     

Screening of intron 1 inversion and three intragenic factor VIII gene polymorphisms in Pakistani hemophilia A families

Indirect linkage analysis using highly informative polymorphic markers is the method of choice for carrier detection of hemophilia A in developing countries because direct DNA or mRNA sequence analysis is manifold costly and difficult than indirect gene tracking. Worldwide populations have revealed marked variation in the informativeness of polymorphic markers because of which each country has to select its own panel of markers for linkage analysis in hemophilia A families. The present study aimed at determining the informativeness of three factor VIII gene polymorphisms [intron 13(CA)n repeats, HindIII and AlwNI] in the Pakistani population. One hundred and forty-three individuals from 32 hemophilia A families and 68 unrelated anonymous females from the general population were screened for these polymorphisms using PCR and RFLP techniques. An inversion in intron 1 of the factor VIII gene causing 2-5% of severe hemophilia A cases was also screened in 128 Pakistani hemophilia A patients. None of the affected individuals carried the intron 1 inversion at least in peripheral blood leucocytes. The informativeness of intron 13 repeats, HindIII and AlwNI was 59.1% (13/22 hemophilia A families revealing five different alleles), 40.6% (13/32 hemophilia A families) and 6.25% (2/32 hemophilia A families), respectively. The cumulative informativeness of intron 13 repeats and HindIII was 63.6% (14/22 hemophilia A families), revealing strong linkage disequilibrium between these two polymorphic markers. These results suggest that there is a need to determine the informativeness of other polymorphic markers of the factor VIII gene to achieve 100% success rate for carrier detection of hemophilia A in Pakistan.     

Analysis of intron 22 inversions of the factor VIII gene in severe hemophilia A: implications for genetic counseling

Intrachromosomal recombinations involving F8A, in intron 22 of the factor VIII gene, and one of two homologous regions 500 kb 5' of the factor VIII gene result in large inversions of DNA at the tip of the X chromosome. The gene is disrupted, causing severe hemophilia A. Two inversions are possible, distal and proximal, depending on which homologous region is involved in the recombination event. A simple Southern blotting technique was used to identify patients and carriers of these inversions. In a group of 85 severe hemophilia A patients, 47% had an inversion, of which 80% were of the distal type. There was no association with restriction fragment length polymorphism (RFLP) haplotypes. The technique has identified a definitive genetic marker in families previously uninformative on RFLP analysis and provided valuable information for genetic counselling information may now be provided for carriers without the need to study intervening family members and the diagnosis of severe hemophilia A made in families with only a nonspecific history of bleeding. Analysis of intron 22 inversion should now be the first-line test for carrier diagnosis and genetic counselling for severe hemophilia A and may be particularly useful when there is no affected male family member or when intervening family members are unavailable for testing.     

Clinical correlates among 49 families with hemophilia A and factor VIII gene inversions

Inversions between a gene A copy within intron 22 of the factor VIII gene and additional copies outside the factor VIII gene were found in 49 families with hemophilia A. Inversion patterns were that of recombination with a distal gene A copy in 34, a proximal copy in 14, and a third (variant) copy in one. Baseline factor VIII clotting activity levels were <1% of normal in 43 and 1% in 6. No inversion was detected in 61 other families whose affected members had ≤1% activity levels nor in 42 families with moderately severe hemophilia A and 2–5% baseline levels. Both high titer and low level alloantibody inhibitors were found in patients with or without an inversion. Of 13 high titer inhibitors, 8 were persistent and 1 of these patients had an inversion. Of 5 that responded to daily factor VIII infusions, 4 were in patients with gene inversions. Of the 49 families with an inversion, the occurrence of hemophilia was isolated in 30 and the mother was a carrier in the 25 in which additional family members were informative. In three of these families with isolated occurrence, the maternal grandmother was a carrier whereas in three others a de novo mutation occurred in the maternal grandfather's factor VIII gene. Screening for gene inversions in patients with severe (or “borderline” severe) hemophilia A provides a direct marker of the mutation in 45% of families. It is useful even if there is no living affected member and in predicting the likely severity of an infant in which there are no reliable baseline clotting activities, including 70% of families with isolated occurrences of hemophilia A. © 1996 Wiley-Liss, Inc.     

Carrier detection and prenatal diagnosis by intron 22 inversion analysis of the factor VIII gene

Summary In approximately 50% of severe haemophilia A patients the mutation is present in the form of a large chromosomal disruption in the factor VIII gene; this disruption is described as an inversion. It results in the physical breakage and separation of exons 1-2 and exons 23-26 of the factor VIII gene.     

Characterization of five partial deletions of the factor VIII gene.

Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, we have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes.     

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).     

Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.     

Development of factor VIII:C antibodies in dogs with hemophilia A (factor VIII:C deficiency)

Classic hemophilia A (factor VIII:C deficiency) was diagnosed in a miniature Schnauzer dog and a breeding program established. Inbreeding and crossbreeding produced 16 hemophilic animals. All were initially treated with canine cryoprecipitate, as required, for sporadic hemorrhagic events. Five animals developed potent antibodies to canine factor VIII:C. All were the offspring of obligate carriers, resulting from the mating of a hemophilic purebred miniature Schnauzer male to a normal female Brittany spaniel. The mean age at first treatment and factor VIII exposure at the time of inhibitor development was 10.3 wk and 286.3 U, respectively. The remaining hemophilic animals have not developed antibodies, despite receiving a mean factor VIII dosage of 1.5 X 10(3) U. This group includes animals derived from a mating between the same purebred miniature Schnauzer hemophilic male and a purebred miniature Schnauzer carrier female. In each case, the antibodies recognize both canine and human but not porcine VIII:C. They are non-precipitating IgG immunoglobulins. Following inhibitor development, infusion of canine cryoprecipitate was hemostatically ineffective and factor VIII:C recovery at 30 min was negligible. Infusion of a concentrate of porcine factor VIII resulted in a correction of the hemostatic defect and optimal factor VIII:C recovery. All animals receiving porcine factor VIII:C subsequently developed antibodies to this protein. The chance occurrence of this complication should facilitate further studies directed at elucidating the pathogenesis and management of hemophilia complicated by the development of antibodies to factor VIII:C.     

Severe haemophilia A in a female resulting from two de novo factor VIII mutations

Summary. A 2-year-old girl is described with severe haemophilia A (factor VIII: C <0-01 units/ml). Both of her parents were phenotypically normal. Cytogenetic analysis on the proband demonstrated an interstitial X chromosome deletion encompassing Xq26-q28. Molecular studies with several polymorphic markers close to and within the factor VIII gene showed that the proband had inherited only the paternal factor VIII gene, indicating that the X chromosome deletion had occurred de novo in the maternal germ line. Further study of the factor VIII gene inherited by the proband from her father showed the presence of a de novo gene inversion mutation (a type 1, distal pattern inversion). Neither parent showed any evidence of the factor VIII inversion in their somatic DNA. The severe haemophilia A documented in this girl is therefore the result of two de novo mutations affecting the factor VIII gene, a maternally derived X chromosome deletion and a paternal factor VIII inversion mutation.     

A novel DNA inversion causing severe hemophilia A

Almost half of all cases of severe hemophilia A are caused by recurrent DNA inversions, which disrupt the factor VIII (FVIII) gene. These inversions generally occur between a region of intron 22 (int22h) and one of two homologous copies of this region, located 300 to 400 kb telomeric to the FVIII gene. They are routinely detected by a Bcl I Southern blot assay in which the sizes of two of the three normal hybridization bands are characteristically altered. However, atypical hybridization patterns have been observed, and this report describes the first detailed analysis of a hemophilia A patient with such a pattern. The abnormal result was found to be caused by a novel FVIII inversion involving an extra copy of int22h from a site only 70 to 200 kb telomeric of the FVIII gene. Polymerase chain reaction (PCR) allowed one of the inversion junctions to be analyzed, showing that the int22h sequence at this inversion junction was truncated. This patient and his novel inversion provide further evidence that int22h is associated with instability in Xq28.     

Immune tolerance induction with recombinant factor VIII in hemophilia A patients with high responding inhibitors

Immune tolerance induction (ITI) eradicates inhibitors in patients with hemophilia A. This study was designed to investigate the success rate of ITI in high-responding inhibitor patients with severe hemophilia A using recombinant factor VIII (rFVIII). Twenty-six patients received different ITI regimens until a normal recovery (>66%) and half-life (>6 h) of infused FVIII was achieved. In order to maximize the chance of success, the initiation of ITI was deferred in the majority of patients until the inhibitor declined to <10 BU. Twenty-two patients (85%) had baseline inhibitor levels <10 BU (median 2.3 BU) when ITI began. Within a median of 6 months, immune tolerance was achieved in 19 of 26 patients (73%) including 12/17 (70%) with intron 22 inversion, 5/7 (71%) with other null mutations and two with small deletion/insertions in the F8 gene. In conclusion, recombinant FVIII induces a high rate of immune tolerance even in carriers of null F8 mutations.