Long-read genome sequencing and variant reanalysis increase diagnostic yield in neurodevelopmental disorders

Abstract

Variant detection from long-read genome sequencing (lrGS) has proven to be considerably more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences "HiFi" technology on 96 short-read-negative probands with rare disease that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, eight of which (8/96, 8.33%) harbored pathogenic or likely pathogenic variants. Newly identified variants were visible in both srGS and lrGS in nine probands (~9.4%) and resulted from changes to interpretation mostly from recent gene-disease association discoveries. Seven cases included variants that were only interpretable in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either: not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

Keywords: Clinical sequencing; LRS Special Issue; Long read sequencing; neurodevelopmental disorder; repeat expansion; structural variation.

Figure 1.. A de novo, 4 Mb paracentric inversion in proband 1, affecting ZBTB20.

A, B. Visualization of a subset of proband and parent reads in IGV at the 5’ (A) and 3’ (B) breakpoints (black arrowheads) indicate a de novo event. C. Alignment of the proband’s assembled paternal contig versus the reference genome supports the inversion. D. Visualization of the inverted region (highlighted in light blue) in the UCSC browser shows the inversion spans 35 protein-coding genes and likely disrupts the ZBTB20 gene (dark blue bar).

Figure 2.. Two ALS2 deletions in trans in Proband 2.

A. Visualization of proband and parent reads in IGV indicate two overlapping deletions in ALS2; a smaller maternal deletion (pink bar) and a larger paternal deletion (blue bar/arrow). Alignment of the proband’s assembled maternal (B) or paternal contig (C) versus the reference genome support the two deletions (red dashed lines).

Figure 3.. Proband 3 has a 4 kb insertion in the 3’ UTR of HCFC1.

A. The proband’s family has a history of X-linked intellectual disability, as the proband (not shown) and two other male relatives (gray squares) are affected. B. Model of the relative length of the insertion in the 3’ UTR. C. The insertion is likely inherited from a heterozygous carrier mother, as indicated by srGS reads.