Federico Bottini

Familial nonrandom inactivation linked to the X inactivation centre in heterozygotes manifesting haemophilia A

A basic tenet of the Lyon hypothesis is that X inactivation occurs randomly with respect to parental origin of the X chromosome. Yet, nonrandom patterns of X inactivation are common – often ascertained in women who manifest recessive X-linked disorders despite being heterozygous for the mutation. Usually, the cause of skewing is cell selection disfavouring one of the cell lineages created by random X inactivation. We have identified a three generation kindred, with three females who have haemophilia A because of extreme skewing of X inactivation. Although they have both normal and mutant factor VIII (FVIII) alleles, only the mutant one is transcribed; and, they share an XIST allele that is never transcribed. The skewing in this case seems to result from an abnormality in the initial choice process, which prevents the chromosome bearing the mutant FVIII allele from being an inactive X.

MLPA assay in F8 gene mutation screening

Besides intron 22 inversion with a reported prevalence of 40–50% in patients with severe haemophilia A (HA) [1], and intron 1 inversion with a reported prevalence of about 2–5% [2], the gene alterations causing HA are spread throughout the gene [3]. Large deletions of one or more gene exons account for about 5% of all cases of severe HA [3]. Over the last few years, we characterized the mutation in a series of patients with severe HA, and traditional PCR failed to amplify one or more exons in four of the 126 patients. As mRNA was not available, we decided to search for further validation. Recently, multiplex ligation-dependent probe amplification (MLPA) has been broadly applied to gene mutation screening to detect exon deletions and duplications [4]. In an ongoing effort to make the technique easier to perform, modifications were made and new MLPA procedures were designed, including the one which contains probes for each of the 26 exons of the F8 gene (SALSA P178 FVIII MLPA kit; MRCAmsterdam, The Netherlands). MLPA evaluation of the multiple amplification relies on the intensity ratio of sample to control, i.e. the relative quantity of each of the PCR products is proportional to the number of copies of the target sequence. Therefore, a ratio proximal or equal to zero indicates deletions of exon/s, a ratio close to 0.5 identifies the presence of the deletion in the heterozygous state. A ratio close to 1.5 identifies the duplication of the exon/s. We present results from a retrospective study that was carried out on patients in whom a deletion or duplication had previously been hypothesized on the basis of PCR alone. MLPA was also applied to the definition of carrier status in the female relatives of the same patients. In fact, the main obstacle to their counselling is the impossibility to demonstrate a heterozygous status because of the amplification of the exon/s that is/are present on the normal X chromosome. Real-time PCR offers the option of carrying out quantification of the amplified material, but simultaneous detection of the gene copy number requires critical consideration of the standardization and reference-curve protocols. In our retrospective study, we demonstrated that carrier status determination via MLPA is straightforward and correct. Multiplex ligation-dependent probe amplification product was performed according to the instructions of the manufacturer. In more detail, 100– 150 ng of genomic DNA were denatured and hybridized with SALSA MLPA probes (60 C overnight). After ligation at 54 C for 15¢, the samples were amplified using a Biometra T personal Thermocycler (Biometra, Göttingen, Germany). One and a half microlitre of PCR products was mixed with 0.3 lL of ROX-500 labelled internal size standard, separated by capillary electrophoresis on a Genetic Analyser 3130 (Applied Biosystem, Foster City, CA, USA), and analysed using the genemapper software (Applied Biosystem). DNA dosage was estimated using coffalyser software (version 6; MRC, Amsterdam, The Netherlands). In the presence of values suggesting deletion/insertion(s) or ambiguous values, MLPA analysis was repeated on independent samples.

Analysis of 18 novel mutations in the factor VIII gene.

Summary. We describe 18 novel mutations, unreported in the Haemophilia A mutation Databases, that have been identified in a cohort of unrelated, Italian patients affected with haemophilia A (HA). Screening of the factor VIII gene (FVIII) was performed using denaturing high‐performance liquid chromatography (DHPLC) and direct sequencing. Eight mutations were characterized as non‐missense alterations, and the remaining 10 were missense mutations. Heterozygosity for the identified mutations was observed in the female relatives of patients belonging to eight families with sporadic cases. In an attempt to understand better the causative effect of the mutations and the clinical variability of the patients, missense mutation consequences were investigated for: (1) the nature of the new amino acid; (2) the location of the substituted amino acid within crystallographic and theoretical models; and (3) the degree of conservation of the native residue in factor VIII (FVIII) protein and FVIII‐related protein family aligned sequences. These research tools have provided evidence that the mutations we describe involve residues that were conserved, at least in FVIII proteins, in all the species we compared.

Insight into molecular changes of the FIX protein in a series of Italian patients with haemophilia B

Summary.  Deficiency or dysfunction of factor IX FIX leads to haemophilia B (HB), an X‐linked, recessive, bleeding disorder. On a molecular basis, HB is due to a heterogeneous spectrum of mutations spread throughout the F9 gene. In several instances, a cause‐effect relation has been elucidated, in others predicted possibilities have been offered by crystallography inspection and by software‐constructed models of the protein. The aim of this study was to contribute to the understanding of HB molecular pathology. The F9 missense mutations we identified in 21 unrelated Italian HB patients by direct sequencing of the whole F9 coding regions were inspected for the causative effect they provoked on the ensuing transcript, and on the protein structure. Each alteration was studied in order to: (i) characterize the defect on the basis of the nature of the mutation; (ii) identify the predicted defect that is induced in the gene and (iii) speculate about the potential, detrimental effects which upset the protein functionality through an idealized FIX model. The resulting data may further contribute to the comprehension of the mechanisms underlying the disease.

Multiplex ligation-dependent probe amplification to detect a large deletion within the von Willebrand gene.

von Willebrand factor (VWF) is an adhesive multimeric glycoprotein, which controls primary haemostasis at sites of vascular trauma or injury by mediating platelet adhesion and aggregation through its subendothelial and platelet interactions. VWF also binds coagulant factor VIII (FVIII:C), thus contributing to its stability and therefore to its function in the generation of fibrin [1]. Qualitative or quantitative VWF defects lead to von Willebrand s disease (VWD), which is the most common congenital bleeding disorder in humans [2]. The von Willebrand gene is located on the short arm of chromosome 12 at p13.3 and is composed of 52 exons (spanning about 178 kb) coding for a protein of 2813 amino acids. A number of distinct functional domains have been identified within the VWF, including regions involved in binding to FVIII, to platelet receptor GP Ib, to platelet GP IIb–IIIa, to components of extracellular matrix such as collagen and heparin, and regions involved in multimerization and dimerization of VWF [3]. A single mutation affecting any one of these domains can lead to the disease. The disorder can be classified into three types, based on a quantitative (types 1 and 3) or qualitative (type 2) defect of the protein. VWD type 3, the most severe form of the disorder, shows markedly reduced or absent VWF in plasma and cellular compartments, as well as reduced plasma FVIII activity. It is transmitted as an autosomal recessive trait and it affects 1.6 per million Italians [2]. In most cases, the disease is caused by two different mutations spread throughout the gene (compound heterozygous), while more rarely, it is caused by a single mutation in a homozygous state. The international database reports that partial/total deletions of one or more exons in the homozygous or heterozygous state are the causative defect in about 10% of the severe forms (http://www.ragtimedesign.com/vwf/mutation/). Traditional PCR fails to identify these patients as the amplification of exon(s) that are present on the normal gene masks the deletion which is present on the gene located on the second chromosome. Recently, a multiplex ligation-dependent probe amplification (MLPA) gene dosage kit has been made available to detect exon deletions or duplications in a variety of genetic diseases. MLPA is a multiplex assay that utilizes specific probes for all the exon sequences of the gene being examined in order to assess the relative copy number of each exon. Each probe is composed of two half-probes that consist of a target specific sequence and of a universal primer sequence to uniformly perform multiplex PCR amplification of all probes. The different length of stuffer sequence linked to each probe allows us to separate the resulting amplification products. These target sequences are unique and present no repeat elements or common polymorphisms. Two recent, specifically designed SALSA MLPA P011-A1 and P012-A1 kits (MRC-Holland, Amsterdam, The Netherlands) were used for VWF screening in our laboratory. The two probe mixes contain probes for the whole VWF gene, with the exception of exon 12, for which no probe is available. Two pairs of probes are present in the kit for the analysis of exon 6 and for exon 28. MLPA analysis was performed according to the manufacturer s instructions. Briefly, 100–150 ng of genomic DNA from patients and normal controls were denatured and then incubated with the P11-A1 and P12-A1 probe mixes, in separate tubes for 16 h at 60 C to hybridize the oligonucleotide probes to the VWF gene exons. After ligation at 54 C for 15 min, 35 cycles of PCR were performed according to the manufacturer s instructions using fluorescently labelled universal primers matching the Correspondence: Maura Acquila, Department of Haematology and Oncology, Thrombosis and Haemostasis Unit, Giannina Gaslini Institute, Largo G. Gaslini n. 5, 16147 Genova, Italy. Tel.: +39 010 563 6277; fax: +39 010 386 204; e-mail: mauraacquila@ospedale-gaslini.ge.it

Mutation analysis impact on the genetic counseling of sporadic hemophilia B families

Previous studies have shown that hemophilia B (HB) is the result of several different mutations, mostly single nucleotide substitutions, in the factor IX (FIX) gene. In order to evaluate the impact of mutation analysis on genetic counseling in sporadic and uninformative HB familial pedigrees, we re‐analyzed by the conformation sensitive gel electrophoresis (CSGE) technique 14 patients, previously studied by restriction fragment length polymorphisms (RFLPs). A single mutation was present within the FIX gene of each patient: 12 mutations were single base substitutions, 1 was a base insertion, and 1 was a four nucleotide deletion; 4/12 mutations have not been described so far. By identifying the detrimental mutations in affected males, carrier status was correctly diagnosed in all the women we studied; 3/12 de novo events were found in maternal meioses with a 25% mutation rate. Identification of the genetic defect was also successfully applied to three prenatal diagnoses.

von willebrand factor multimer composition is modified following oral methionine load in women with thrombosis, but not in healthy women

Hyperhomocysteinemia is associated with an increased risk of venous and arterial thrombosis, probably by inducing endothelial damage. Von Willebrand factor (VWF) is an endothelial marker protein. It is a plasma multimeric molecule that plays a thrombophilic role. Our purpose was to investigate VWF changes in patients with thrombosis following oral methionine load. We evaluated homocysteine levels and VWF parameters (plasma levels, activity, proteolysis fragments, and multimer composition) before and after methionine load in 42 women with venous or arterial thrombosis and in 36 healthy women. Methionine load induced mild hyperhomocysteinemia in 10 patients and two controls. No changes in VWF levels and activity were observed, but an increased amount of VWF proteolysis fragments was found post-load in patients and controls. VWF multimer composition was unaffected in controls, while a decrease of the largest VWF multimers was found in women with thrombosis. Homocysteine levels inversely correlated with the amount of the largest multimers in hyperhomocysteinemic patients. Large VWF molecules were probably released from endothelial cells following load, and rapidly cleaved by the specific VWF-cleaving protease. VWF proteolysis was enhanced in mild hyperhomocysteinemic patients, thus leading to downregulation of VWF size to smaller multimers.

Germ-line origin of intron 1 inversion in two haemophilia A families.

Summary.  Factor VIII gene inversion of intron 1 has recently been reported to be the mutation responsible for haemophilia A in about 5% of severe cases. In our series of patients, which is made up of 77 Italian cases negative for intron 22 inversion, the mutation was found in three sporadic and in one familial patients, with an overall frequency of 5.2%. The carrier status of the patients’ female relatives was assessed by mutation analysis and showed that only two‐thirds of cases could be considered truly sporadic. The germ‐line origin of the mutation was investigated in the two sporadic families by haplotype analysis on genomic DNA of the patients’ maternal grandparents. These studies indicated that both mutation events had occurred in the germ cell lines of the patients’ healthy grandfather, suggesting that, as already demonstrated for the inversion of intron 22, the male germ cell line is more susceptible to the intrachromosome recombination which leads to the inversion of intron 1.

A new strategy for prenatal diagnosis in a sporadic haemophilia B family: A RAPID PRENATAL DIAGNOSIS IN A HAEMOPHILIA B FAMILY

Although the quality of life for haemophiliacs has clearly improved in the last few years, haemophilia still remains a serious disorder justifying prenatal diagnosis (PD) and, if necessary, termination. Because chorionic villus sampling (CVS) is performed in the first trimester of pregnancy, an increasing number of carriers are interested in this test. It has been shown that waiting for the results is particularly distressing for pregnant women, therefore decreasing the diagnostic procedure time can be psychologically helpful. Here we report on PD in a sporadic haemophilia B family based on the direct identification of the pathogenic mutation in a CVS taken at the 12th gestational week. In order to hasten the results, we recovered DNA from a single villus fragment boiled in water and used it directly for PCR reaction. Conformation‐sensitive gel electrophoresis (CSGE) was used to detect the mutation in the haemophilia carrier and in the foetus. This approach allowed us to obtain a diagnosis within 24 h of CVS, thus avoiding the long‐term psychological effects on the pregnant woman.

Identification of mutations in exon 14 including five novelties in 13 Italian patients with haemophilia A

Haemophilia A (HA) is the most common X-linked, recessive bleeding disorder which affects from one in 5000 to one in 10 000 males. The disorder is caused by mutations within the FVIII gene (FVIII), which is located at Xq28 and consists of a 9 kb coding region. Other than the inversion of intron 22, reported with a prevalence of 40–50% of patients with severe HA [1], and the inversion of intron 1, reported with a prevalence of about 2–5% [2–4], the gene alterations causing HA are spread all over the gene [5] and include mostly point mutations, small insertions and deletions. In a series of HA, unrelated, Italian patients, referred to our centre for genetic counselling, we have identified 13 mutations in exon 14. Briefly, following informed consent, genomic DNA was extracted from citratated peripheral blood samples, and amplified for FVIII coding regions, promoter and splicing sequences, as described elsewhere [6,7]. Amplified fragments showing an abnormal peak pattern in denaturing high-performance liquid chromatography (DHPLC Helix Varian system; Varian Analytical Instruments, Palo Alto, CA, USA) were sequenced using a fluorescent ABI Prism BigDye terminator kit (Applied Biosystem, Warrington, UK), and the mutations were characterised. Each mutation was confirmed on a second, independent, amplified polymerase chain reaction (PCR) sample. For each of the novel mutations, 50 control samples were analysed and evidence for polymorphisms was ruled out. As shown in Table 1, six of 13 mutations were nonsense alterations and were found to be associated with a severe phenotype, consistently with the premature truncation of the factor VIII protein in the reading translation. Seven mutations were frameshift, all but one was associated with the severe form of the disease. History of neutralizing antibodies to FVIII was negative in all patients.