The Detection of Large Deletions of PROS1 - a Common Cause of Protein S Deficiency
Authors: Wheeler Ruth B , Cutler Jacqueline A , Mitchell Mike 
Published: April 2020

Abstract
Protein S is a vitamin-K dependent plasma glycoprotein which regulates coagulation by acting as a non-enzymatic co-factor for activated protein C during the inactivation of FVa and FVIIIa. Protein S circulates in the plasma as either free protein S or in association with C4b binding protein but only free protein S has anticoagulant properties. Protein S deficiency is an autosomal dominant disorder associated with an increased risk of venous thrombosis. The PROS1 gene is located on the long arm of chromosome 3 near the centromere, in close association with a highly homologous pseudogene PROSP. More than 200 PROS1 mutations have been described to date, but a large cohort of patients exists in whom no mutations have been detected. Several recent studies have suggested that large deletions of PROS1 are common in protein S deficient patients and should be considered when conventional methodologies fail to identify the causative mutation. Therefore there is a requirement for robust and reliable tests capable of rapidly detecting and defining deletions which can be used in a diagnostic setting. We have developed an approach which combines haplotype analysis at several SNPs and simple sequence repeats located throughout the PROS1 genomic sequence which runs in parallel with conventional mutation analysis, followed by confirmation of DNA copy number by quantitative PCR using an ABI7500 Real Time PCR System and more recently multiplex ligation-dependent probe amplification (MLPA) using the SALSA MLPA P112 Kit (MRC Holland). We have used this approach to identify and define large PROS1 deletions in three of six patients referred with protein S deficiency, and in whom mutations had not been detected. Patient 1 presented with recurrent thrombosis and was found to have a severe type I protein S deficiency. PROS1 sequence was normal, but analysis of parental DNA at Pro626 identified the presence of a null allele. Real-time PCR and MLPA revealed that one whole copy of the gene was deleted in this patient. A homozygous mutation in exon 2 was identified in patient 2 who presented with severe protein S deficiency. Analysis of DNA from his son, who also presented with protein S deficiency, showed exon 2 to be normal indicating that this sequence change was hemizygous. A partial deletion involving exons 1 to 8 was subsequently detected in this family. No phenotypic data was available for family 3, which comprised two sisters, but our combined approach identified a deletion of over 50Kb involving the 5′UTR and exons 1 and 2. This highlights the usefulness of this methodology for mutation screening when phenotypic data is unavailable, which is often the case when patients are on continuous anticoagulant therapy. None of the remaining three patients had a family history of protein S deficiency and phenotypic data was inconclusive. Analysis failed to identify any genetic abnormalities in these patients. Our analyses confirm that deletions of PROS1 are a common cause of thrombosis. Further when deletions are not detected using this methodology the information may be useful as supporting evidence for clinicians when reaching a diagnostic decision as to the cause of thrombosis. In summary our approach offers a rapid and reliable approach to the detection of large protein S deletions which would otherwise be missed by routine technologies.