High Throughput Sequencing in 3449 Patients with Bleeding and Platelet Disorders: Novel Gene Discovery and Robust Diagnosis

Background

Bleeding symptoms are common and reported at some point by 25% of the population. Laboratory investigations frequently fail to provide an explanation for the symptoms or to identify those at significant risk of future bleeding. A similar problem is presented by patients with congenital thrombocytopenia which often cannot be distinguished from acquired disorders and making investigation of platelet function impractical. The broad heterogeneity of phenotypes associated with established disorders further complicates diagnosis. For these reasons, we have developed High Throughput Sequencing (HTS) approaches to investigate patients with undiagnosed bleeding and platelet disorders (BPDs) through the ThromboGenomics 79 gene panel HTS (TG) test and by whole genome sequencing (WGS).

Methods

BPD cases were recruited in 3 broad categories: Group 1: 1321 cases suspected to have a defect in one of the 79 known BPD genes (ie genes known to harbour variants responsible for a BPD); Group 2: 212 cases being prepared for elective surgery with self-reported bleeding symptoms; and Group 3: 1916 cases with a suspected inherited BPD, not thought to be caused by a variant in one of the 79 known BPD genes. DNA samples from the first two groups were sequenced by the TG test and from the third group by WGS as part of the first 35,000 samples for the 100,000 Genomes Project.

In total, 3449 DNA samples from BPD patients have been analysed by HTS. Laboratory and clinical phenotypes were recorded using Human Phenotype Ontology (HPO) terms. Variants were called and prioritised based on minor allele frequency, predicted impact and presence in the Human Gene Mutation Database and those variants present in the 79 known BPD genes were reviewed by a multi-disciplinary team (MDT) where pathogenicity was assigned to variants using ACGS criteria (Clearly pathogenic [CPV], Likely pathogenic [LPV] and Variant of unknown significance [VUS]).

Results

A mean of 6.6 variants were assessed at MDT for each case. In Group 1 pathogenic variants were observed in 713 of 1321 (54%) of cases and >40% were novel and labelled as LPV; ~20% of variants discussed were designated VUS. In sharp contrast, pathogenic variants were only identified in 6 of 212 (2.8%) Group 2 cases. In Group 3, pathogenic variants were identified in 106 of 1916 (12%) cases. In >300 cases with non-syndromic thrombocytopenia, CPV or LPV were identified in ~30%. In contrast, in 131 cases with a significant bleeding disorder but no identified laboratory platelet or coagulation abnormality causal variants were observed in only 5 cases.

Conclusion

These results demonstrate the success of HTS approaches in providing a genetic diagnosis for patients with well-defined inherited platelet or coagulation defects. In addition, 23 novel BPD genes were identified by analysis of the WGS data from Group 3 cases, including NBEAL2, RBM8A, SRC, DIAPH1, TPM4, ABCC4 and KDSR. Furthermore extensive replication was demonstrated in nearly 100 Group 3 cases for recent BPD gene discoveries by other groups (C6ORF25, CYCS, RNU4ATAC, STIM1, RASGRP2, ETV6, ACTN1). All MDT-reviewed variants and appended HPO terms are shared in ClinVar, supporting the international effort to improve reference catalogues, aligned to ASH's Precision Medicine initiative.

Discussion

HTS has been particularly successful for thrombocytopenia, but the yield for bleeding disorders without numerical platelet or coagulation defects is low. Our results, and the observations by others are compatible with the notion that the genetic architecture of unresolved BPD cases is extremely diverse. Continuing efforts such as the 100,000 Genomes Project with sharing and transparency of data across projects will enable better interpretation of variants and the discovery of novel genes. Finally, we are annotating the non-coding space with data from recently identified GWAS variants for blood cell traits, BLUEPRINT and ROADMAP epigenome marks, and eQTLs for blood cell traits to interpret the regulatory regions of the genome and identify non-coding variants causing BPD and other unresolved rare diseases of the blood.