Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: Identification of seven novel mutations and three novel, apparently neutral, variants

Abstract

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non-isotopic Single-Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single-stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5'-upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single-strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C->G, L-27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co-segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (-296C->T, 182G->C and T57S), were identified in a normal control, two type I/III and two type III PS-deficient pedigrees.

Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S–C4b binding protein complex

“…It has been suggested that they are phenotypic variants of the same genetic disorder, as both phenotypes were found to coexist in the same families. 123,148,[155][156][157] In one of these families, deficient individuals were subsequently found to carry the same causative mutation, and the change in phenotype (type I to type III) was shown to be due to an age-related increase in TPS levels. 140 The mutation present in this family (Gly295Val) was later shown to completely abrogate PS expression from the affected allele.…”

A novel rare c. -39C>T mutation in thePROS15’UTR causing PS deficiency by creating a new upstream translation initiation codon and inhibiting the production of the natural protein

“…Some intronic splice variants (Choi et al , 2011; Menezes et al , 2017; Wang et al , 2019) and structural variants (Hurtado et al , 2009; Lind-Halldén et al , 2012; Seo et al , 2014) have also been reported. Note that, compared to exonic variations, very few variations have been described in the promoter region of the PROS1 gene (Espinosa-Parrilla et al , 2000; Hurtado et al , 2008; Li et al , 2019; Sanda et al , 2007; Tang et al , 2013) and even fewer have been functionally characterized. To our knowledge, the c.-168C>T is the sole PROS1 promoter variation that has been experimentally demonstrated to cause inherited PSD by affecting the core binding site of Sp1 transcription factor (Sanda et al , 2007).…”

Co‐segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1

“…Nevertheless, screening for PROS1 gene mutations has been performed in a number of studies. The proportion of cases where no mutations are detected varies widely between studies [10,17,[23][24][25][26][27][28][29][30][31][32][33]. When all cases are pooled together, PROS1 gene mutations are not detected in 47% of PS deficient families.…”