Long-read genome sequencing identifies causal structural variation in a Mendelian disease

Abstract

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions > 50 bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.

Figure 1. Clinical history and three-dimensional transthoracic echocardiography of patient with multiple neoplasia including cardiac myxomata

(A) Patient narrative. VT = ventricular tachycardia (B) A 2 × 3 cm myxoma is seen in the left ventricular outflow tract (white arrow). (C) The 2 × 3 cm myxoma is seen from another perspective (lower left, white arrow). A 5 × 4 cm myxoma is seen in the right atrium (lower right, white arrow).

Figure 2. Heterozygous deletion in PRKAR1A

(A) PacBio long reads identify a heterozygous 2,184 bp deletion that includes the first coding exon of PRKAR1A. Two of four reads at the locus support the deletion. (B) Sanger sequencing confirms the deletion. The forward (YH_479426-1073) and reverse (YH_479426-1074) sequences from a representative amplicon agree to the base pair with the higher quality PacBio read, PacBio_53019216. (C) Illumina short reads support the heterozygous deletion variant through a drop in read coverage and clipped reads at the deletion breakpoints.