Targeted long-read sequencing identifies and characterizes structural variants in cases of inherited platelet disorders.

Background: Genetic diagnosis of inherited platelet disorders (IPDs) is mainly performed by high-throughput sequencing (HTS). These short-read-based sequencing methods sometimes fail to characterize the genetics of the disease.

Objectives: To evaluate nanopore long-read DNA sequencing for characterization of structural variants (SVs) in patients with IPDs.

Methods: Four patients with a clinical and laboratory diagnosis of Glanzmann thrombasthenia (GT) (P1 and P2) and Hermansky-Pudlak syndrome (HPS) (P3 and P4) in whom HTS missed the underlying molecular cause were included. DNA was analyzed by both standard HTS and nanopore sequencing on a MinION device (Oxford Nanopore Technologies) after enrichment of DNA spanning regions covering GT and HPS genes.

Results: In patients with GT, HTS identified only 1 heterozygous ITGB3 splice variant c.2301+1G>C in P2. In patients with HPS, a homozygous deletion in HPS5 was suspected in P3, and 2 heterozygous HPS3 variants, c.2464C>T (p.Arg822∗) and a deletion affecting 2 exons, were reported in P4. Nanopore sequencing revealed a complex SV affecting exons 2 to 6 in ITGB3 (deletion-inversion-duplication) in homozygosity in P1 and compound heterozygosity with the splice variant in P2. In the 2 patients with HPS, nanopore defined the length of the SVs, which were characterized at nucleotide resolution. This allowed the identification of repetitive Alu elements at the breakpoints and the design of specific polymerase chain reactions for family screening.

Conclusion: The nanopore technology overcomes the limitations of standard short-read sequencing techniques in SV characterization. Using nanopore, we characterized novel defects in ITGB3, HPS5, and HPS3, highlighting the utility of long-read sequencing as an additional diagnostic tool in IPDs.