Whole Exome Sequencing in Evaluation of Thrombophilia: A Novel 33-Gene Panel
Authors: Lee Eun-Ju , Dykas Daniel , Bale Allen , Cromwell Caroline , Parker Terri L , Halene Stephanie , Burns Adrienne , Yao Xiaopan , Lee Alfred I 
Published: October 2019

Abstract
Introduction:
Venous thromboembolism (VTE) occurs with an incidence of 1-2 per 1000 individuals per year. Approximately 10-20% of patients with VTE have a heritable thrombophilia involving one of five known genes: Factor V, prothrombin (PT), antithrombin (AT), protein C (PC), and protein S (PS). Significant variability of laboratory functional assays as well as fluctuating plasma levels of AT, PC and PS lead not only to delay in initial thrombophilia screening but also to multiple rounds of costly testing. Whole exome sequencing (WES) is a potentially useful diagnostic tool for inherited thrombophilias as it avoids dependence on laboratory and situational variation in protein levels. We compiled a panel of 33 genes involved in thrombosis with a goal of investigating the diagnostic yield and cost of WES in comparison to traditional thrombophilia testing.
Methods:
Since January of 2014, we have been performing WES in patients with a personal and family history of VTE seen at Yale New Haven Hospital. Thus far, 18 such patients have had a complete WES analysis. Data regarding patient demographics, number and type of VTE events, family/surgical history, medical co-morbidities, and traditional laboratory testing for inherited thrombophilias was recorded. Costs of each test were determined based on the amount billed to insurance. WES focusing on 33 genes involved in thrombosis was performed and analyzed by the DNA Diagnostic Lab at the Yale School of Medicine. Positive WES testing was defined as identification of a pathogenic variant in a gene known to be associated with thrombophilia, found at a frequency consistent with frequency of the disease, and with evidence that the variant predisposes to thrombosis. Positive laboratory testing was defined as any test that led to an unequivocal diagnosis of Factor V Leiden, PT mutation, homozygous MTHFR mutation with hyperhomocysteinemia, or a deficiency in AT, PC, or PS.
Results:
All 18 patients (7 male, 11 female) were included in the final analysis. Median age at first VTE was 35.5 years (range, 14-78 years); median number of independent VTE events was 2 (range, 1-9). WES with our 33-gene thrombophilia panel was positive in 11 of 18 (61.1%) patients, while traditional laboratory testing was positive in only 2 of 16 (12.5%) cases (Table 1). There were no statistically significant differences in clinical characteristics between those patients with positive findings on WES versus those without. Identified variants included those in genes with well known roles in thrombosis (SERPINC1, PROS1, F5), and in genes with emerging data regarding thrombosis (HABP2, SERPINA10, SERPIND1). Two patients identified on WES as having PS deficiency, one with AT deficiency, and one with a non-Leiden Factor V mutation had laboratory testing that was either normal or uninterpretable. The total cost of WES at our institution was $1935.00; by comparison, among 16 patients who underwent laboratory testing, median cost of laboratory testing was $2892.70 (range, $406.90-$11419.80), and the cost of laboratory testing exceeded WES in 13 patients.
Conclusions:
WES using our 33-gene thrombophilia panel has higher diagnostic yield and is more cost effective than traditional thrombophilia testing. With increasing availability and declining cost, this thrombophilia gene panel has the potential to truly transform thrombophilia testing. Further investigation of the diagnostic power and phenotypic correlation of identified mutations is in progress.
Table 1. Mutations detected by the 33 gene thrombophilia panel Gender Age (yrs) Lab testing result WES result M 40 Negative SERPINA10 (Q384R) heterozygous M 67 Negative SERPINC1 (S426W) heterozygous M 30 Negative PROS1 (Y234C) heterozygous F 34 Negative HABP2 (G508E) heterozygous F 56 APC resistance F5 (R506Q) heterozygous F 27 F2 20210 G>A heterozygous F2 20210G>A heterozygous; vWF P2063S heterozygous F 37 Negative SERPIND1 (R468C) heterozygous F 48 Negative F5 (T915S) heterozygous M 20 Negative PROS1 (P76L) heterozygous M 55 Negative SERPINA10 (21_23delCCT ) heterozygous F 78 Negative HABP2 (G534E) heterozygous F 49 Negative Negative F 41 Negative Negative M 32 Negative Negative F 64 Negative Negative M 42 Negative Negative F 51 Negative Negative F 28 Negative Negative

Disclosures
No relevant conflicts of interest to declare.