Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families. (Blood. 2011;117(13): 3684-3691)

Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wildtype recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecularweight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that iso- Introductionvon Willebrand factor (VWF) is a large multimeric glycoprotein that has 2 important roles in hemostasis. These are stabilizing factor VIII (FVIII), by acting as its carrier protein in the circulation, and attaching activated platelets to the subendothelium via binding to the GpIb receptor on platelets and to collagen in the subendothelial matrix. Enhancement of the platelet-subendothelium interaction is vital at sites of vascular injury during conditions of high shear stress. VWF binds to collagen via 2 sites: the A3 domain (residues 1683-1874) contains the main site for fibrillar collagen types I and III found within perivascular connective tissue, 1,2 and a second site in the A1 domain (residues 1260-1471) binds nonfibrillar collagen type VI within the subendothelial matrix. 3,4 However, the A1 domain can also bind collagen types I and III, and the relative importance of the 2 domains has been extensively but inconclusively investigated. 5,6 In all cases, it appears that affinity of VWF for collagen is heavily dependent on the presence of VWF multimers of high or ultrahigh molecular weight. 7 Type 2 von Willebrand disease (VWD) is characterized by a qualitative defect in VWF and is diagnosed by demonstration of a discrepancy between circulating plasma levels of VWF and its functional activity. In addition to measuring GpIb␣-dependent function using ristocetin cofactor activity (VWF:RCo), it has been recommended that collagen-binding activity (VWF:CB) is analyzed in the subclassification of type 2 VWD. 8 A single defect in VWF collagen binding in association with normal multimeric structure has previously been reported. 9 In this report, we describe the phenotypic and genotypic characterization of 3 families with bleeding symptoms...

Factor XI (FXI) functions in blood coagulation. FXI is composed of four apple (Ap) domains and a serine protease (SP) domain. Deficiency of FXI leads to an injury-related bleeding disorder, which is remarkable for the lack of correlation between bleeding symptoms and FXI coagulant activity (FXI:C). The number of mutations previously reported in our interactive web database (http://www.FactorXI.org) is now significantly increased to 183 through our new patient studies and from literature surveys. Eight novel missense mutations give a total of 120 throughout the FXI gene (F11). The most abundant defects in FXI are revealed to be those from low-protein plasma levels (Type I: CRM-) that originate from protein misfolding, rather than from functional defects (Type II: CRM+). A total of 70 Ap missense mutations were analysed using a consensus Ap domain structure generated from the FXI dimer crystal structure. This showed that all parts of the Ap domain were affected. The 47 SP missense mutations were also distributed throughout the SP domain structure. The periphery of the Ap beta-sheet structure is sensitive to structural perturbation caused by residue changes throughout the Ap domain, yet this beta-sheet is crucial for FXI dimer formation. Residues located at the Ap4:Ap4 interface in the dimer are much less directly involved. We conclude that the abundance of Type I defects in FXI results from the sensitivity of the Ap domain folding to residue changes within this, and discuss how structural knowledge of the mutations improves our understanding of FXI deficiencies.

Investigation of three families with von Willebrand disease showed that haemorrhagic symptoms were associated with disproportionately reduced collagen binding activity whilst Ristocetin co-factor activity was commensurate with antigen and multimeric analysis was normal. Genetic analysis revealed heterozygosity for two novel mutations in two of the families: W1745C in exon 30 and S1783A in exon 31. In the third family the affected individuals were heterozygous for a previously-described mutation: S1731T in exon 30 but two unaffected individuals also carried this mutation. All three mutations lie in the A3 domain containing the main collagen binding site in VWF. In patients’ samples VWF:CB activity was measured using human type I and type III collagen. Patients heterozygous for W1745C and S1731T showed a reduction in binding to both collagens but more marked reduction in binding to type III collagen. Heterozygosity for S1731T resulted in mild impairment of type I collagen binding but normal binding to type III collagen. Site-directed mutagenesis was used to generate vectors containing the three mutations (S1731T, W1745C and S1783A) and also one containing a W1745A mutation. Mutated VWF was expressed in HEK293T cells both singly and in co-transfection with a wild-type VWF (wtVWF) vector. All VWF mutants were expressed at a similar rate to wtVWF. Multimeric analysis demonstrated that all the mutants had a similar multimeric structure compared to recombinant wtVWF. However recombinant-wtVWF (wtVWF) had a lower collagen binding to VWF antigen ratio (CB:Ag) compared to plasma VWF (0.39 type I collagen and 0.45 type III collagen vs >0.7 for plasma VWF). This is most likely due to the slight shift towards lower molecule weight multimers seen with recombinant VWF. CB:Ag ratios for the recombinant VWF showed the same pattern of binding to collagen type I and III as the clinical samples. The W1745A mutant demonstrated a similar CB:Ag ratio to W1745C. Kinetic analysis of binding to type I collagen demonstrated that W1745C, W1745A and S1783A did not bind and that S1731T bound with significantly less affinity compared to wtVWF (KD,app 27.1 ± 0.5nM and 7.3 ± 0.8nM respectively). Analysis of binding to type III collagen demonstrated that W1745C and W1745A both bound with ~ 8-fold reduced affinity (KD,app 16 ± 2.6nM and 21.3 ± 6.3nM) but wtVWF and S1731T bound with similar affinity, (KD,app 2.0 ± 0.1nM and 3.7 ± 0.85nM respectively). Analysis of the crystal structure of the VWF A3 domain showed that W1745 may interact with Y1780 and we noted the mutation Y1780A has also been shown to significantly reduce collagen binding. Measurement of free thiols present in VWF demonstrated that the new cysteine residue in W1745C is not involved in disulphide bond formation. These results indicate that it is the loss of W1745 rather than the creation of a new cysteine residue that is responsible for the loss of collagen binding activity. We therefore hypothesised that W1745 and Y1780 participate in an internal aromatic interaction that helps to maintain the structural configuration of A3. We sought confirmation by expressing another mutant; W1745F, replacing the tryptophan with another aromatic amino acid. As predicted this did not significantly affect collagen binding. In conclusion, our findings demonstrate that type 2 VWD may be arise from mutations in A3 causing abnormal collagen binding without other functional defects or abnormalities in multimer formation. This type of VWD may be under-recognised unless laboratories measure binding to both types I and III collagen. Mutations in A3 yield insights into the structural requirements for collagen binding may have differential effects on binding to collagen types I and III and can result in variable clinical phenotypes. Some mutations may not be consistently associated with bleeding symptoms.

The molecular basis of haemophilia B is heterogeneous and many mutations of the Factor IX (FIX) gene have been characterised. Using the allele-specific arrayed primer extension (AS-APEX) technology, we have designed a FIX array to simultaneously analyse 69 mutations found in British, Thai and Chinese patients. This technology overcomes the problem of multiple reverse dot-blot analysis and has a 100% accuracy in the detection of both affected subjects and carriers in families with known mutations. In seven unknown mutations from Thailand, the array could detect the specific mutation in five and in the remainders the normal primer at specific spots failed to extend due to a mutation a few nucleotides upstream, thus allowing their identification. Hence this FIX array can detect 53% of the 2891 mutation entries in the FIX database. Each of the microarray slide can be used for three different test samples and would be useful for carrier testing for common mutations and prenatal diagnosis. It is simpler and more cost effective than genome sequencing and would be particularly useful in laboratories with limited technical capabilities.