Search

Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes ( GRIN2A , GRIN2B , IL20RB , IL26 , PER3 , PITX2 , and PPARGC1A ) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six ( CAMK2G , GRIN2A , GRIN2B , N1RD1 , PER3 , PPARGC1A ) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs ( n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants ( GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.

Objective: Functional polymorphisms contributing to coagulation factor levels are preferential markers for association studies aimed at identifying prothrombic genetic components., Methods and Results: Factor V (FV) microsatellite genotypes were found to be associated with FV levels (P=0.003). Single nucleotide polymorphisms analysis and sequencing of the promoter and of coding regions identified two polymorphisms (Met2120Thr, Asp2194Gly) present in 20% of the population (n=1013) that are responsible for genotype-phenotype associations. The effect of the Met2120Thr polymorphism, both in plasma (mean reduction of FV level in the heterozygous condition: 25%) and in recombinant FV studies (34% reduction), was comparable to that of the Asp2194Gly change (20% and 34%, respectively). The study of 10 subjects with a rare genotype indicated that the Asp2194Gly substitution is the functional determinant of the reduced FV levels associated with the FVHR2 haplotype. Among Leiden carriers, the doubly heterozygous condition for FV2120Thr was found to be associated with a significantly increased activated protein-C resistance (APCR) (P<0.05), and the doubly heterozygous condition for FV2194Gly was found to be more frequent (P=0.009) in symptomatic than in asymptomatic subjects., Conclusions: Extensive analysis of FV polymorphisms indicated that changes in the C2 domain modulate FV levels and might increase APCR and thrombotic risk in FV Leiden carriers through a pseudohomozygous mechanism.

The role of factor V (FV) mutations in activated protein C (APC) resistance and FV deficiency is well established. We report on the identification of a highly polymorphic (AT)n microsatellite marker in the FV gene, which represents an informative tool for the investigation of the origin and evolution of pathologically relevant FV genetic components. A high number of different microsatellite alleles were found to be associated with FV R506Q and FV H1299R, two single-origin mutations. An example of the use of the microsatellite marker in family studies of thrombophilia and FV deficiency is also provided.

Background and Objectives: Factor V (FV) deficiency is a rare bleeding disorder whose molecular bases are poorly characterized. We have recently described a FV missense mutation (Y1702C) predicting reduced FV levels in a thrombophilic patient and in a healthy individual. The aim of the present work was to assess the prevalence of the FV Y1702C mutation among subjects with FV deficiency., Design and Methods: Carriership of the FV Y1702C mutation was tested in 8 patients with severe FV deficiency (FV:C <8%), in 16 individuals with asymptomatic partial FV deficiency (mean FV:C 38.0%, SD 11.6%) and in 9 patients with pseudo-homozygous APC-resistance (mean FV:C 46.2%, SD 3.6%). An AccI-restriction protocol was employed for rapid mutation screening., Results: The FV Y1702C mutation was detected in two unrelated patients with unmeasurable FV levels (one being homozygous and the other doubly heterozygous for a still unknown mutation) and in one subject with partial FV deficiency (FV:C 30%). A striking difference in bleeding phenotype was observed between the homozygous patient and her asymptomatic brother with the same FV genotype. A multi-point FV haplotype analysis was performed in all unrelated carriers of the FV Y1702C mutation. Three haplotypes were found to underlie the mutation in different individuals, suggesting that it might have arisen independently more than once., Interpretation and Conclusions: FV Y1702C is a common cause of FV deficiency in the Italian population and might be a recurrent mutation.

Two novel polymorphisms were identified in the factor V gene by direct sequencing of intronic areas. One of them, located in intron 9, is the marker closest to the Leiden mutation ever described, whereas the other, in intron 16, displays a rare allele invariantly associated to the mutation. Allele-specific amplification protocols were designed to perform extensive screenings for both polymorphic sites. The new markers were used in combination with six previously described polymorphisms to define specific factor V gene haplotypes. Haplotype investigations in 506Q homozygous thrombotic patients and normal controls showed the presence of a single haplotype underlying the factor V Leiden mutation in Mediterranean populations (among which Greek Cypriots, where the R506Q mutation is particularly frequent) and Indians. When traced in the absence of the Leiden mutation, the background haplotype was found to be present and roughly as frequent as the mutation itself in these populations. These findings indicate a single mutational event, that probably occurred outside Europe, as the cause of the Leiden mutation and provide a powerful tool to investigate its evolutionary history.