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Congenital factor XIII (FXIII) deficiency is a rare autosomal recessive disorder, usually attributed to a defect in the FXIII A subunit, whose genetic basis has been studied in a number of cases. We describe here the genetic variations found in two unrelated patients with FXIII deficiency. Both patients, under prophylactic substitution with FXIII concentrate, showed low plasma FXIII A subunit antigen levels with undetectable A subunit antigen in the platelets and normal plasma B antigen levels, which indicate that the defects are present in the A subunit of the molecule. Both probands were heterozygous for a previously reported G-->A transversion in exon 8 of the FXIII A subunit gene (Arg326Gln substitution). Proband 1 was also heterozygous for a novel G-->T transversion in exon 7, which predicts a Val316Phe substitution. Two of her sons were heterozygous for this mutation and showed low FXIII activity and FXIII A subunit antigen levels. Val316 is a well-conserved amino acid among the transglutaminase family, located within the core domain, close to the Cys314 member of the catalytic triad. Proband 2 had a unique 2-bp (TT) insertion in one of the alleles within or adjacent to the -7 to -20 T tail of intron A. This insertion was not found in 50 healthy individuals, which supports this being the second mutation in this patient.

Hypoprothrombinemia is a rare hereditary coagulation defect characterized by low levels of biologically active prothrombin. In this paper we report the laboratory and genetic analysis of a patient with a severe hypoprothrombinemia and some of her relatives. Laboratory analysis showed very low levels of prothrombin antigen. Molecular analysis of the prothrombin genes of the patient resulted in the identification of two novel sequence variations in heterozygous state, a 20079 G to A transition, which predicts a Trp 569-->Stop mutation, and a 1261C-->G change within intron B near the acceptor splice site. A cosegregation of prothrombin deficiency in family members with the two genetic defects was observed.

The standard approach for the molecular genetic analysis of protein C deficiency, polymerase chain reaction (PCR) amplification followed by direct sequencing, although very accurate, is time-consuming. The aim of this study is to investigate the usefulness of a simplified, time-saving screening method for the detection of protein C mutations consisting of the combination of multiplex PCR amplifications using the same primers that were designed for sequencing, followed by single-strand DNA conformation polymorphism (SSCP) electrophoresis analysis performed with one set of conditions. The study was designed in two phases. First, we tested six known point mutations located in different exons of the protein C gene by SSCP. Second, we prospectively studied nine patients with protein C deficiency type I using SSCP as the first screening technique. All the exons were amplified with a common PCR protocol, either as single fragments or as multiplex combinations of several of them. In the retrospective study, three out of the six point mutations were visible as a band shift: 40 T-->G (exon 2), 1432 C-->T (exon 3) and 7253 C-->T (exon 8). In the prospective analysis SSCP detected three different mutations. These mutations were: 6128 T-->C (exon 7), 6216 C-->T (exon 7) and in two probands 8631 C-->T (exon 9). In the five remaining patients we identified only two different mutations by direct sequencing: 6246 G-->A (exon 7) in two patients and 8589 G-->A (exon 9) in four patients. In summary, the results from both studies show that only 60% of all mutations can be detected using this simplified method. It also suggests that a multiple set of conditions, smaller PCR fragments, or both, should be used in order to achieve a sensitivity comparable to sequencing.

The abnormal prothrombin gene of an Italian patient with a severe bleeding tendency and hypoprothrombinemia was selected for study and compared with the prothrombin genes of healthy controls. All the coding and their flanking regions and the 5'- and 3'-UT regions of the prothrombin gene were screened by analyzing the nucleotide sequence of the corresponding PCR products. The patient was found to be heterozygous for two novel point mutations: one at nucleotide 4251 in exon 6, which changes the codon for cysteine-138 (TGC) in the kringle 1 domain to that for tyrosine (TAC), and one at nucleotide 8812 in exon 10, which results in the replacement of tryptophan-357 (TGG) by cysteine (TGT) in the catalytic domain. Her mother was heterozygous for the Cys-138 Tyr mutation and her father heterozygous for the Trp-357 Cys mutation. Several other sequence variations were identified in the prothrombin genes from control individuals. Only the variations at nucleotide 4203 and 10253 could be established as polymorphisms.

We described molecular genetic studies of 15 patients with protein S deficiency type I (i.e. reduced total protein S antigen). All the exons of the PROS 1 gene were analyzed both by PCR and direct sequencing in all 15 probands. This analysis led to the identification of point mutations affecting eight individuals. One of these mutations (codon-25, insertion of T) has been described previously in a Dutch pedigree. The other mutations are novel and all are located in exons that code for the protein S domain that is homologous to the steroid hormone binding globulins. They include two amino acid replacements (one individual with 340 Gly--> Val, and two individuals with 467 Val --> Gly), and four frameshift mutations due to either one bp deletions (in codon 261 deletion of T and in codon 267 deletion of G) or insertions (in codon 565 insertion T and after codon 578 insertions of C). Studies performed in six families (totalling 43 subjects) showed cosegregation of the genetic abnormality with reduced plasma protein S levels, and provided genetic evidence for a heterozygous protein S deficiency in 25 of them. The yield of mutations in this study (53%) confirms that the percentage of protein S deficient cases in which a point mutation is found remains low.

The molecular basis of hereditary type I and type II protein C deficiency was studied in a panel of 14 unrelated Austrian families. By direct sequencing of the nine exons and their splice junctions sequence alterations were found in one of the protein C alleles in all but one subject. In twelve subjects a single alteration was found whereas in one subject one of the protein C alleles carried two sequence abnormalities. Whenever DNA from family members was available (11 of the 14 cases) cosegregation of the protein C deficiency with the mutation was observed. In contrast to what has been found previously in a panel of Dutch patients with hereditary protein C deficiency, none of the 14 mutations occurred in more than one family. Only two of the genetic defects (157Arg-->Stop and 178Arg-->Gln) have been found previously in other geographic locations. These data confirm the large genetic heterogeneity of protein C deficiency.