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Haemophilia A is the most common inherited bleeding disorder caused by defects in the F8C gene that encodes coagulation factor VIII. This X-linked recessive disorder occurs in approximately 1:5000 males. Haemophilia A is diagnosed based on normal prothrombin time, altered activated partial thromboplastin time and reduced factor VIII activity in plasma. Carrier females are usually asymptomatic and can be identified only by molecular analysis. The most frequent mutations in F8C are intron 22 and 1 inversions, which occur in approximately 50% and 5% of patients, respectively, with a severe phenotype. Large gene deletions are observed in approximately 5% of alleles from patients with severe haemophilia A. The remaining severe cases and all moderate and mild cases result from numerous point mutations and small insertions/deletions, which are de novo mutations in one-third of cases. Thus, molecular diagnosis of carrier status and prenatal diagnosis in families without intron 22 or 1 inversions is based on scanning techniques or gene sequencing. When the disease-causing mutation cannot be identified, molecular diagnosis is performed by linkage analysis of several DNA polymorphic markers linked to F8C. Given the clinical heterogeneity among haemophilic patients, many groups, including our own, have examined the relationships between prothrombotic gene variants and haemophilic phenotype to investigate whether prothrombotic gene variants modify clinical expression of the disease.

Thanks to its typical expression, haemophilia can be identified in writings from the second century AD. Haemophilia B, an X-linked recessive bleeding disorder due to factor IX (FIX) deficiency, has an incidence of about 1:30,000 live male births. The factor 9 (F9) gene was mapped in 1984 on Xq27.1. Haemophilia is diagnosed from prothrombin time, activated partial thromboplastin time, and FIX levels. Carrier females are usually asymptomatic and must be identified only with molecular analysis. Linkage analysis of F9 polymorphisms is rapid and inexpensive but limited by non-informative families, recombinant events, and the high incidence of germline mutations; thus, various procedures have been used for the direct scan of F9 mutations. We set up a novel denaturing high performance liquid chromatographic procedure to scan the F9 gene. This rapid, reproducible procedure detected F9 mutations in 100% of a preliminary cohort of 18 haemophilia B patients. Parallel to the development of more efficient diagnostic tools, the life expectancy and reproductive fitness of haemophilic patients have greatly improved and will continue to improve thanks to the use of less immunogenic recombinant FIX. Hopefully, new approaches based on gene therapy now being evaluated in clinical trials will revolutionise haemophilia B treatment.

Haemophilia A (HA) is an X-linked recessive haemorrhagic disorder caused by a deficiency of coagulation factor VIII. Disease causative mutations are heterogeneous and spread all over the F8 gene sequence, with the exception of intron 22 inversion occurring in about 50% of severe patients. To define the specific mutational repertoire and mutation detection rate, we analysed F8 gene, by polymerase chain reaction and direct sequencing, in 128 unrelated patients from Southern Italy with severe (n = 108), moderate (n = 9) or mild (n = 11) HA. We identified 120 mutations, including 64 cases of intron 22 inversion (53.3%), three of intron 1 inversion (2.5%), one large deletion (0,8%) and 52 point mutations (43.3%). In particular, 19/120 were novel and 7/52 point mutations (13.5%) occurred at CpG sites. We also investigated eight prothrombotic genetic variants in a subgroup of 74 severe HA patients to evaluate their possible protective effect on the severity of clinical expression. Methylenetetrahydrofolate reductase A1298C and plasminogen activator inhibitor-1 4G variants recurred more frequently in HA patients with a less-severe phenotype. Clinical impact of these findings needs large-scale studies to further define the role of these prothrombotic variants as possible modifier factors of HA phenotype.

Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome is a rare autosomal dominant ectodermal dysplasia syndrome. It is caused by heterozygous mutations in the TP63 gene encoding a transcriptional factor member of the p53 family. Mutations in TP63 gene, mainly missense in exons 13 and 14 encoding the sterile alpha motif (SAM) and the transactivation inhibitory (TI) domains, account for 99% of patients. Of these, ≥ 70% are de novo mutations, present in the affected patient, but not in parents nor in healthy siblings. However, when a mutation firstly appears, it is not possible to differentiate between: i) a sporadic mutation, and ii) a germline mosaicism in parents. In this latter case, the risk of further affected children cannot be excluded. We describe a case of mosaicism for a novel TP63 mutation identified in two sisters, both affected by AEC syndrome. Both carried the heterozygous c.1568T>C substitution in exon 13 of TP63 resulting in a p.L523P change in the SAM domain of the protein. Analyses of DNA from blood cells (from both parents), seminal fluid (from the father) and maternal cells (buccal, vaginal and cervical) did not reveal the mutation, suggesting that the mosaicism may involve a very low percentage of cells from one of the parents (very low grade somatic mosaicism) or, more likely, be a maternal germline mosaicism. Mosaicism must be considered for the assessment of recurrence risk during genetic counseling in AEC syndrome, and preimplant/prenatal diagnosis should be offered to all couples, even when the mutation is apparently de novo.

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PD for CF should be performed in reference laboratories equipped for gene scanning and linkage analysis, with a multidisciplinary staff able to offer counselling to couples during all phases of PD.

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