Abstract
Objective Duchenne muscular dystrophy (DMD) is caused by pathogenic variants in the dystrophin gene (DMD). Hypermethylated CGG expansions within DIP2B 5′ UTR are associated with an intellectual development disorder. Here, we demonstrate the diagnostic utility of genomic short-read sequencing (SRS) and transcriptome sequencing to identify a novel DMD structural variant (SV) and a DIP2B CGG expansion in a patient with DMD for whom conventional diagnostic testing failed to yield a genetic diagnosis.
Methods We performed genomic SRS, skeletal muscle transcriptome sequencing, and targeted programmable long-read sequencing (LRS).
Results The proband had a typical DMD clinical presentation, autism spectrum disorder (ASD), and dystrophinopathy on muscle biopsy. Transcriptome analysis identified 6 aberrantly expressed genes; DMD and DIP2B were the strongest underexpression and overexpression outliers, respectively. Genomic SRS identified a 216 kb paracentric inversion (NC_000023.11: g.33162217-33378800) overlapping 2 DMD promoters. ExpansionHunter indicated an expansion of 109 CGG repeats within the 5′ UTR of DIP2B. Targeted genomic LRS confirmed the SV and genotyped the DIP2B repeat expansion as 270 CGG repeats.
Discussion Here, transcriptome data heavily guided genomic analysis to resolve a complex DMD inversion and a DIP2B repeat expansion. Longitudinal follow-up will be important for clarifying the clinical significance of the DIP2B genotype.
Duchenne muscular dystrophy (DMD) is a rare X-linked recessive muscular dystrophy caused by pathogenic loss-of-function variants in DMD (MIM: 300377). Some patients with DMD also demonstrate cognitive impairment.1 FRA12A is a distinct intellectual developmental disorder caused by a heterozygous expanded CGG repeat in the 5′ UTR of the disco-interacting protein 2 homolog B gene (DIP2B; MIM: 611379) on chromosome 12q13.
Here, we report a patient with DMD and mild intellectual disability harboring a novel DMD structural variant (SV) and a DIP2B short tandem repeat (STR) expansion. We exemplify how a combination of sequencing approaches, including transcriptome sequencing, genomic short-read sequencing (SRS), and targeted long-read sequencing (LRS), can be leveraged to resolve complex genetic variants.
Methods
Genomic SRS
Proband DNA was run on version 3 of a custom-designed muscle disease–targeted gene panel at Diagnostic Genomics, PathWest, as described previously.2
Illumina genome-wide SRS was performed at the Broad Institute Center for Mendelian Genomics (CMG), as described previously.3 SV and SNV analysis was performed using seqr (seqr.broadinstitute.org), which leverages Manta (Illumina).4 Genotyping of the DIP2B STR locus expansion was performed using ExpansionHunter.5
Genomic LRS and Methylation Profiling
LRS was performed at the Garvan Institute using Oxford Nanopore Technologies (ONT) “ReadUntil” selective sequencing to target the loci of interest (DMD and DIP2B), as demonstrated recently.6
Transcriptome Sequencing
Skeletal muscle transcriptome sequencing was performed at the CMG, as previously described.7 Aberrant expression events were detected using OUTRIDER.8 Skeletal muscle transcriptome sequencing samples from the CMG Rare Disease Cohort were used as controls (n = 172).
Highly expressed junctions from all 803 GTEx v3 muscle samples were used as controls for generating the Sashimi plot. The junction-spanning read counts were normalized to represent the average spanning read count per sample.
Results
The proband had a typical DMD clinical presentation with delayed motor development; he first walked at 18–20 months of age. He could walk fast but never ran and required carrying because of fatigue. He had frequent falls and used a Gowers' maneuver to raise from the floor. He had ASD with poor socialization at school, no friends, and engaged in solo play. His IQ and language were within normal limits. Fragile X and microarray testing were normal. There was no relevant family history.
Skeletal muscle immunoperoxidase staining revealed a complete absence of dystrophin (Figure 1, A and B), consistent with dystrophinopathy. Hematoxylin and eosin staining showed variation in myofiber size and some active degeneration (Figure 1C). No SNVs, indels, or SVs in DMD were detected by the initial analysis of genomic SRS data.
Immunoperoxidase staining reveals the absence of DYS1 (DYS2 and DYS3 protein not shown) in the patient biopsy section (A), relative to an unaffected control (B). (C) Hematoxylin and eosin shows increased variation in myofiber size (10–90 µm) and some active degeneration. All images are at 100× magnification.
OUTRIDER analysis of muscle transcriptome data identified 6 aberrantly expressed genes, including DMD (Z score = −7.43, adjusted p value = 1.66 × 10−5; Figure 2A). Sashimi plots showed retained junctions in the 3′ region for the Dp71 and Dp40 isoforms and some 5′ exon 1 and intron 1 reads (Figure 2B). Manual investigation of genomic SRS data in the 5′ DMD preserved region revealed split reads in a pattern suggestive of a 216 kb paracentric inversion (NC_000023.11:g.33162217-33378800; eFigure 1, links.lww.com/NXG/A587). Analysis of the breakpoint junction reads identified microhomology domains within 2 transposons, a telomeric SINE, and a centromeric DNA/hAT-Charlie element, suggestive of a mechanism of microhomology-mediated break-induced repair. The inversion overlaps promoters for the (cortical) DMD isoform (Dp427c) and the full-length muscle isoform (Dp427m). No common inversions are seen in the DMD locus in gnomAD-SV (v2.1).
(A) Volcano plot from OUTRIDER analysis showing aberrant gene expression detected in a patient. (B) Sashimi plots of the patient (top) and normalized splice junctions from 803 muscle control samples from GTEx v3 (bottom). The major skeletal muscle DMD isoforms were obtained from Refseq.
DIP2B overexpression was the strongest outlier (Z score = 6.14, adjusted p value = 5.00 × 10−7: Figure 2A). Manual inspection of the DIP2B locus in genomic SRS data revealed a drop in coverage within the 5′ UTR (eFigure 1, links.lww.com/NXG/A587). STR genotyping indicated a repeat genotype of 109 (80-186)/7 (7-7). This was the largest expansion in DIP2B in all samples within the CMG and RGP Rare Disease Cohorts (n = 3,245). Only 3 samples in gnomAD (n = 18,511) had genotypes in this range.
Targeted LRS6 confirmed the presence of the large inversion spanning the promoters of Dp427c and Dp427m and the repeat expansion in DIP2B (Figures 3A, 3B). The expanded DIP2B allele was estimated at 270 × CGG repeats. There was no evidence of hypermethylation on the expanded haplotype, which was predominantly unmethylated at the DIP2B promoter in whole blood (Figure 3C). The methylation status of DIP2B was not evaluated in other tissues such as brain.
(A) Alignments showing a large inversion in DMD spanning the promoters of Dp427c and Dp427m (reverse strand gene). The left- and right-side breakpoints are shown magnified in the black boxes. (B) Genotyping (left) and methylation profiling (right) of the DIP2B 5′ UTR STR expansion locus. A heterozygous expansion (270 × CGG) was detected in the patient but absent in healthy controls (n = 10). There was no evidence of DNA hypermethylation in the patient profile, compared with controls. b.p., base pairs; hap, haplotype; UTR, untranslated region.
Discussion
Here, we describe the utility of transcriptome combined with genomic SRS and LRS to identify a complex DMD inversion.
Transcriptome sequencing is becoming increasingly important for molecular diagnosis of patients with Duchenne and Becker muscular dystrophy who have dystrophinopathy based on muscle biopsy but negative on genomic testing.3,9 SVs are increasingly recognized in DMD and can be overlooked without transcriptome data,3 contributing to the 1.5%–7% of DMD variants that are not detectable through genomic analysis.9 Intronic variants leading to pseudoexon inclusion also evade conventional DNA testing.9
Genomic LRS offers the capacity to detect pathogenic substitutions, copy number changes, complex rearrangements, repeat expansions, and methylation differences, all within a single data source. These complex variants are challenging to identify through clinical genetic testing, which typically rely on SRS, such as exome sequencing or gene panels. The versatility of LRS has made it an active area of investigation in muscular dystrophy; recent studies have leveraged LRS to identify complex variants in patients with DMD previously lacking a genetic diagnosis, including intronic repeat expansions,10 large inversions,11 and deep intronic splice-altering SNVs.11 In addition, LRS can be used to study transcriptome dynamics involved in the pathology of muscular dystrophy12; LRS is well adapted to analyze repetitive elements, can clearly detect exon connectivity, and shows whole isoforms with high confidence.12
The clinical significance of DIP2B overexpression in our patient with DMD remains unknown. DIP2B is associated with FRA12A, a folate-sensitive chromosomal fragile site associated with an autosomal dominant intellectual developmental disorder.13 The molecular basis of FRA12A is an expansion of the CGG repeat locus located in the CpG island at the 5′ UTR of DIP2B.13. Methylation of the expanded allele and a ∼50% reduction in DIP2B expression were previously identified in all patients with DIP2B-related intellectual disability.13 The estimated sizes of the previously described expansions were 273–306 × CGG.13 An unaffected carrier of a smaller and unmethylated expansion showed increased levels of DIP2B mRNA; thus, repeat elongation may behave as a transcriptional enhancer.13 These prior findings suggest that the unmethylated 270 × CGG expansion and DIP2B transcript overexpression do not contribute to our patient's clinical phenotype, although we note that this was not assessed in brain tissue.
DMD can be associated with cognitive involvement, including autism spectrum disorders.14 It is difficult to discern whether the cognitive phenotype in our single patient is due to the disruption to the Dp427c isoform (which has previously been shown to cause CNS comorbidities1) or the DIP2B overexpression. It is also possible that both genotypes and other genetic variants create a blended cognitive phenotype. There are no available data on the impact of DIP2B overexpression. FMR1 premutation carriers who have excessive FMR1 transcript levels develop fragile X–associated tremor/ataxia syndrome.15 An analogous phenomenon could apply to DIP2B. Furthermore, the techniques used previously to size DIP2B repeats (PCR and Southern blotting)13 are imprecise; therefore, an accurate pathogenic repeat threshold for DIP2B expansions remains undetermined, adding to the difficulty of interpreting the clinical significance of this variant in our patient. Longitudinal follow-up will be important to determine whether the patient may have mild symptoms of this repeat or may develop a later-onset neurodegenerative phenotype. Additional cases are needed to discern whether there is a phenotype associated with DIP2B overexpression. Further long-read studies are required to explore the pathogenic range of DIP2B expansions.
Study Funding
This project was supported by an Australian NHMRC Ideas Grant (APP2002640) and NHMRC Investigator Fellowship (APP2007769) to G.R. Short-read sequencing and analysis were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and were funded by the National Human Genome Research Institute (NHGRI), the National Eye Institute, and the National Heart, Lung, and Blood Institute grant UM1HG008900 and NHGRI U01HG011755.
Disclosure
The authors have no disclosures relevant to the manuscript. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG.
Appendix Authors
Footnotes
The Article Processing Charge was funded by the authors.
Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG.
Submitted and externally peer reviewed. The handling editor was Associate Editor Margherita Milone, MD, PhD.
- Received September 20, 2022.
- Accepted in final form January 27, 2023.
- Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
References
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