High Frequency of Genetic Variants Influencing Diagnosis and Bleeding Pattern in Patients with Presumed Primary Immune Mediated Thrombocytopenia (ITP)
Authors: Ljungström Elmfors Einar , Rasmussen Andreas Ørslev , Andersson Nadine G. , Martensson Annika , Leinoe Eva Birgitte , Rossing Maria , Zetterberg Eva 
Published: December 2023

Introduction
Immune mediated thrombocytopenia (ITP) is a platelet disorder in which the immune system attacks and destroys the body's own platelets. Mechanisms leading to low platelet count in ITP are multifactorial, involving both increased peripheral platelet destruction and decreased platelet production. This is caused by autoantibodies targeting megakaryocytes and platelet-specific glycoproteins, as well as cytotoxic T cells directly acting on platelets. Although antibodies in some cases can be found in patients with ITP, there is no well-established diagnostic test and the diagnosis is thus, one of exclusion. While most cases of ITP resolve within 3 months with or without treatment, some patients do not respond to conventional treatment and develop a refractory ITP. It is likely that in the group of patients diagnosed with refractory ITP, some does not have an immune mediated thrombocytopenia, but other causes of low counts (PLC)s. With the development of high throughput sequencing and the increasing knowledge of inherited bleeding disorders, the possibility to identify the underlying cause of disease in these patients is steadily growing. In ITP, there is a high degree of interindividual differences in bleeding rates, where some patients have no bleeding symptoms despite of unmeasurable PLC, whereas other patients show severe bleeding symptoms at PLC &gt; 30 x10 9 /L. The underlying etiology of these discrepancies is currently unknown and complicates the clinical care of these patients. We hypothesize that genetic defects influence the bleeding pattern in true ITP patients as well as being one of the underlying causes of thrombocytopenia in patients misdiagnosed with “refractory ITP”. The aim of this pilot study was to investigate whether upfront genetic screening of patients suspected of ITP could be a valuable diagnostic tool to determine presence of inherited thrombocytopenia, secondary thrombocytopenia and genetic variants that might affect bleeding pattern.
Method
Patients were included after informed consent following the declaration of Helsinki. We performed whole genome sequencing (WGS) in patients diagnosed with presumed ITP at the Skåne University Hospital, Sweden. Whole genome sequencing was performed at the Center for Genomic Medicine at Rigshospitalet, Copenhagen. An in-silico gene panel consisting of 113 genes known to be associated with bleeding disorders as well as 86 genes associated with ITP (Using the VarSeq gene prioritisation algorithm phorank) was analysed and pathogenicity was classified according to the ACMG/AMP guidelines. Patients' medical records were examined, laboratory work up was performed and clinical data regarding demographics and information regarding bleeding was collected. A final evaluation of susceptible variants took place at multiple disciplinary team conferences.
Results
Twenty-one patients were enrolled (9 women, 12 men, 17 were adults and 4 children). Mean age of adult patients was 50,5 years and mean age of paediatric patients was 11,5 years. Out of 21 patients, 7 were shown to have genetic variants with relevance to their disease. All variants were found in adult patients. Two patients had variants related to inherited thrombocytopenia (X-linked thrombocytopenia(WAS) and monoallelic Bernard-Soulier Syndrome(GP1BB)), 4 had variants in genes related to secondary ITP (3 with variants associated with common variable immunodeficiency (NFKB1, TNFRSF13B) and 1 with systemic lupus erythematosus (TREX1)) and 1 had a variant associated with platelet dysfunction (platelet delta storage pool deficiency (UNC13D)). Only the variant associated with X-linked thrombocytopenia was previously described (class 5) whereas the remaining 6 variants were initially classified as variants of unknown significance (VUS). Functional work-up and co-segregation studies indicated them as probably pathogenic (class 4) but confirming studies are still pending.
Conclusion
In this pilot study, 33% of patients with presumed primary ITP had variants with relevance to their clinical phenotype, and we were able to diagnose 1 patient with an inherited bleeding disorder (X-linked thrombocytopenia). Whole genome sequencing seems to be a useful diagnostic tool in the work up of patients with thrombocytopenia where ITP is suspected to ensure the diagnosis and could also add important information when assessing bleeding risk in individual patients.