Combining optical genome mapping and RNA-seq for structural variants detection and interpretation in unsolved neurodevelopmental disorders

Abstract

Background: Structural variations (SVs) are key genetic contributors to neurodevelopmental disorders (NDDs). Exome sequencing (ES), the current first-line tool for genetic testing of NDDs, falls short in SVs detection. This diagnostic gap is being actively addressed by new methods such as optical genome mapping (OGM).

Methods: This study evaluated the utility of combining OGM and RNA-seq in the detection and interpretation of SVs in ES-negative NDDs. OGM was performed in 43 patients with NDDs with inconclusive ES results. Candidate SVs were selected based on disease association and pathogenicity evaluation, and further validated or reconstructed by alternative methods, including long-read sequencing for a complex rearrangement event. RNA-Seq was performed on blood samples from patients with candidate SVs to facilitate interpretation of pathogenicity.

Results: OGM detected four candidate SVs, and RNA-seq confirmed the pathogenicity of three SVs in the patient cohort. This combined approach solved three cases-two cases with de novo SVs in genes associated with autosomal dominant NDDs, including a deletion encompassing the promoter and 5'UTR of MBD5 and an intragenic duplication of PAFAH1B1, and a third case possessing an intragenic duplication in trans with a pathogenic single-nucleotide variant of PLA2G6, associated with autosomal recessive NDDs. The expression alteration of the affected genes and the tandem positioning of two intragenic duplications were confirmed by RNA-seq. In the fourth case, OGM detected a complex rearrangement involving chromosomes 2 and 6, much more complex than the de novo t(2:6)(q13;q15) indicated by conventional cytogenetic analysis. Reconstruction showed that 17 segments of 6q15 spanning 9.3 Mb were disarranged and joined 2q11.2, with four breakpoints detected in the 5' and 3' non-coding region of the NDD-associated gene SYNCRIP. RNA-seq revealed largely preserved SYNCRIP expression, leaving the pathogenicity of this complex rearrangement event uncertain.

Conclusions: SVs in ES-negative NDDs can be identified by OGM, which is particularly useful for SVs in non-coding regions not covered by ES. OGM helps to construct complex SVs and provides information on the location and orientation of duplications, which is crucial for pathogenicity interpretation. The integration of RNA-seq facilitates the interpretation of the functional consequences of SVs at the transcriptional level. These findings demonstrate the utility and feasibility of combining OGM and RNA-seq in ES-negative cases with NDDs.

Keywords: Neurodevelopmental disorders; Optical genome mapping; RNA-seq; Structural variants.

Fig. 1

Overview of SVs filter combining OGM and RNA-seq. a Workflow of OGM analysis. b The diagnostic rate of combining OGM and RNA-seq (3/43, 6.9%)

Fig. 2

Characterization of a de novo 134-kb heterozygous deletion partially involving MBD5 and ORC4 in P40. a OGM showed a deletion of 5′-untranslated exons of MBD5 (ogm[GRCh38] 2q23.1(147,966,774_148,100,811) × 1). b UCSC Genome Bowser showed that the deletion included the 5′UTR and promoter of MBD5. c The deletion in MBD5 was not captured by the ES capture kit (xGen Exome Research Panel v1.0, IDT). d RNA-seq confirmed significantly reduced expression of MBD5 together with the nearby gene ORC4 in the patient’s blood compared to controls.  e The normalized read count of P40 was the lowest in the cohort tested

Fig. 3

A de novo heterozygous 9.5-kb intragenic tandem duplication from exon 3 to exon 5 within the PAFAH1B1 gene in P4. a OGM of the PAFAH1B1 locus showed an intragenic insertion. b Integrative Genomics Viewer (IGV) of ES data showed a copy number gain of exons 3–5 in PAFAH1B1. c RNA-seq showed an abnormal fusion junction involving exon 5 to exon 3 along with abnormal splicing events

Fig. 4

Characterization of a SV within the PLA2G6 gene in P2. a OGM showing an approximately 19 kb insertion within PLA2G6 gene. b IGV of the ES data showed an overlooked copy number gain of exon 6–12. c RNA-seq showed an abnormal back-spliced fusion junction between exon 6 and exon 12

Fig. 5

Schematic diagram of der(2) and der(6), and disruption of the NDD-associated gene SYNCRIP. a A schematic diagram illustrating the pattern of recombination after the DNA double strands break in chromosomes 2 and 6 by combining OGM and LRWGS analysis. b Integrative Genomics Viewer showed that the SYNCRIP gene was disrupted by four breakpoints