Optical genome mapping identifies a balanced inversion disrupting DMD in a patient with Duchenne muscular dystrophy

Abstract

Background

Duchenne muscular dystrophy (DMD) is a severe disorder that primarily affects males due to its X-linked recessive inheritance. It is caused by pathogenic variants of the DMD gene, most commonly exonic deletions, duplications, or point mutations. Current routine genetic testing methods, including next-generation sequencing and multiplex ligation-dependent probe amplification, can identify pathogenic DMD variants in over 90% of clinically diagnosed patients. However, in rare cases, a molecular diagnosis cannot be established using routine methods.

Case presentation

We describe a follow-up genetic analysis, based on karyotyping and optical genome mapping (OGM), of a patient with clinically diagnosed DMD who initially had negative results in extensive routine genetic testing. Karyotyping revealed a paracentric X-chromosomal inversion with estimated breakpoints at p22.31 and p21.2. OGM fine-mapped this alteration as inv(X)(p22.2p21.1) and confirmed its pathogenicity by identifying the proximal breakpoint within intron 41 of DMD, thereby disrupting the gene and providing a definitive molecular genetic diagnosis.

Conclusions

Current results further underscore the important role of chromosomal inversions as causal in a subset of DMD patients who remain without a molecular diagnosis after routine testing. It also demonstrates the utility of OGM in providing detailed, gene-level insights into cytogenetic abnormalities observed in the diagnostics of neuromuscular disorders.

Background

Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy characterized primarily by progressive skeletal and cardiac muscle degeneration due to lack of dystrophin protein in muscles [1]. About one-third of patients also exhibit intellectual disability [2]. DMD is inherited in an X-linked recessive manner and caused by pathogenic variants in the DMD gene, located on the short arm of the X chromosome (p21.2–p21.1). The spectrum of pathogenic DMD variants consists mainly of copy number variants (CNVs), including multi-exonic deletions (~60–70%) and duplications (~5–15%), as well as single nucleotide variants (SNVs) or small insertions and deletions (indels) (~20%) [1, 3, 4]. Deep intronic variants causing splicing defects and complex gene-disrupting structural variants (SVs) such as translocations and inversions are very rare (~1–2%) [3, 4]. Pathogenic DMD variants are identified in over 90% of clinically diagnosed patients using current routine genetic testing, which primarily consists of next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) analysis [4, 5]. However, a small subset of patients remain without confirmed genetic diagnosis after these tests, likely due to deep-intronic variants or complex SVs [4, 6]. This highlights the importance of additional genetic testing that can detect alterations that are missed by conventional diagnostic methods.

Here, we describe a follow-up genetic analysis of a patient with clinically diagnosed DMD who remained genetically unsolved after extensive diagnostic testing. We applied optical genome mapping (OGM), a novel high-resolution technique for SV detection [7], with the aim of achieving molecular confirmation of the diagnosis and expanding knowledge of cryptic SVs affecting DMD that may escape the detection by routine analysis methods.

Case presentation

The proband was a 3-year-old boy, the first-born child of the family, when he came to pediatric neurologic evaluation because of developmental delay. He had learned to walk independently at the age of 2 years, and his habitus was hypotonic. His creatine kinase (CK) was elevated up to 25 478 U/l, which together with motor delay, clumsiness, positive Gowers´ sign and mild calf hypertrophy raised suspicion of DMD. He also had a delay in cognitive and language skills and social interaction. His mother had an elevated CK level of 2 135 U/l. She reported some muscle stiffness in her legs and a history of learning difficulties at school. The proband’s maternal uncle was unaffected and none of the family members had a diagnosis of a neuromuscular disease.

Genetic analysis

Following the initial clinical suspicion of DMD, MLPA analysis of the DMD gene was performed to detect exonic CNVs. Following negative MLPA results, extensive genetic testing was carried out, including (1) targeted NGS of the DMD gene, (2) fragile-X testing, (3) SNP-based CGH array, and (4) trio-exome sequencing of the index patient and parents, all of which were negative. Additionally, a chromosome G-banding analysis was performed, which revealed a seemingly balanced inversion with estimated breakpoints at p22.31 and at p21.2 (Fig. 1A). A fluorescence in situ hybridization (FISH) analysis by X-chromosome painting was performed to rule out the presence of translocations involving X-chromosome (Fig. 1B). Subsequent chromosome G-banding analysis confirmed that the mother of the index patient was a carrier of the same inversion.

Fig. 1.

Identification of a paracentric X-chromosomal inversion. (A) G-banding analysis of the proband’s karyotype revealed a paracentric inversion with estimated breakpoints at p22.31 and p21.2. (B) FISH-analysis with whole chromosome X painting (red) and X and Y chromosome p-arm telomere region probes (green) ruled out the presence of X-chromosomal translocations

Although the estimated proximal breakpoint of the inversion resided in the same cytoband as 3’ end of DMD (p21.2), karyotyping could not conclusively confirm its disruption. Therefore, to achieve high-resolution characterization of the inversion breakpoints, OGM analysis was performed as described previously [8]. This refined the inversion as inv(X)(p22.2p21.1) with proximal breakpoint located within intron 41 of DMD. The size of the inversion was 18.9 Mbp and the distal breakpoint resided within an intergenic region at p22.2 (Fig. 2). These findings provided a molecular diagnosis for the patient and enabled subsequent carriership testing in the family, showing that the maternal grandmother of the index was not a carrier of the inversion (Fig. 3).

Fig. 2.

Optical genome mapping analysis of the index patient’s X-chromosome p-arm revealed a balanced inversion and its breakpoints at high-resolution, characterized as inv(X)(p22.2p21.1). The proximal breakpoint was located at chrX:32,330,404~32,337,248 (Hg38/GRCh38), residing within intron 41 of DMD (NM_004006.3). The distal breakpoint was located at an intergenic region chrX:13,447,650 ~ 13,483,855. Tilde (~) represents uncertainty of the exact breakpoint positions in the OGM analysis

Fig. 3.

Pedigree. The proband (indicated by an arrow) was diagnosed at age 3 years. The carrier mother (II-2) had elevated creatine kinase level (2135 U/L) and reported learning difficulties in childhood and mild muscular symptoms. The maternal grandmother (I-2) is a confirmed non-carrier. The maternal uncle (II-4) and maternal grandfather (I-1) are asymptomatic adults and did not undergo carrier testing. The maternal aunt (II-3) is asymptomatic at adult age and was offered carrier testing with karyotyping and OGM due to the risk of parental germline mosaicism, but the results are still pending

Based on our findings, we reviewed the literature for similar reported cases by performing a proximity search using terms “DMD inversion“[Title/Abstract:~25] in PubMed and “DMD inversion” [all:~25] in PubMed Central. This search identified several studies published over the past decade in which balanced DMD inversions were detected using long-read technologies, including long-read sequencing and OGM, after routine genetic tests yielded negative results (summarized in Table 1) [9–25].

Table 1.

Balanced inversions reported over the past decade in DMD cases with negative routine testing

Ref.	Year	Inversion	Size	Breakpoint 1	Breakpoint 2	Detection method
Current	2025	inv(X)(p22.2p21.1)	18.9 Mb	Intergenic	DMD intron 41	KT/OGM
[23]	2025	inv(X)(p22.11p21.1)	8.7 Mb	Intergenic	DMD intron 44	OGM/LRS
[23]	2025	inv(X)(p22.13p21.1)	14.7 Mb	NHS	DMD intron 44	LRS
[9]	2025	inv(X)(p21.1q26.2)	99 Mb	DMD intron 44	Intergenic	OGM
[10]	2024	inv(X)(p21.1q21.1)	50.7 Mb	DMD intron 55	Intergenic	srWGS
[11]	2024	inv(X)(p21.1q27.3)	110.8 Mb	DMD intron 47	Intergenic	LRS
[11]	2024	inv(X)(p21.3p21.1)	6.3 Mb	Intergenic	DMD intron 41	LRS
[12]	2024	Intragenic inversion	77 kb	DMD intron 67	DMD intron 62	LRS
[13]	2024	Intragenic inversion	297 kb	DMD intron 5	DMD intron 1	LRS
[20]	2023	inv(X)(p21.2p21.1)	1.3 Mb	Intergenic	DMD intron 45	OGM/LRS
[14]	2023	Intragenic inversion	216 kb	DMD intron 1	Intergenic	srWGS/LRS
[21]	2023	inv(X)(p21.1q21.31)	55 Mb	DMD intron 2	Intergenic	OGM/LRS
[21]	2023	inv(X)(p21.1q25)	95 Mb	DMD intron 16	Intergenic	LRS
[15]	2023	inv(X)(p21.1q26.2)	99.8 Mb	DMD intron 47	HS6ST2	KT/FISH/LRS
[16]	2022	inv(X)(p21.3p21.1)	5.9 Mb	Intergenic	DMD intron 2	LRS
[18]	2022	inv(X)(p22.12p21.1)	10 Mb	Intergenic	DMD intron 2	srWGS
[18]	2022	inv(X)(p22.2p21.1)	15 Mb	GRPR	DMD intron 54	srWGS
[24]	2022	Inv(X)(p22.31p21.2)	23 Mb	VCX2-VCX3B	DMD intron 60	RNAseq
[17]	2022	inv(X)(p21.1q13.3)	43 Mb	DMD intron 44	Intergenic	srWGS/KT
[19]	2020	Intragenic inversion	982 kb	DMD intron 51	DMD intron 7	LRS
[22]	2017	inv(X)(p21.3p21.1)	5.1 Mb	ENSG00000228933	DMD intron 38	srWGS/OGM
[25]	2017	inv(X)(p21.2q28)	119.8 Mb	DMD intron 17	Intergenic	RNAseq/srWGS
[25]	2017	inv(X)(21.1)	2.7 Mb	DMD intron 18	Intergenic	RNAseq/srWGS

FISH; fluorescence in situ hybridization, KT; karyotyping, LRS; long-read sequencing, OGM; optical genome mapping, srWGS; short-read whole-genome sequencing

Discussion and conclusions

A wide spectrum of pathogenic DMD variants have been reported, most frequently exonic deletions, followed by duplications, point mutations and small indels [1]. In the majority of DMD cases, these variants are detected using routine diagnostic methods, generally MLPA analysis and/or NGS [4]. However, a small fraction of cases harbor genomic rearrangements, such as balanced inversions, that escape these standard methods, leaving these patients without a definite genetic diagnosis [4, 6].

In this case report, we describe a 3-year-old patient with strong clinical suspicion of DMD in whom standard genetic testing was negative, but karyotyping and subsequent OGM revealed a paracentric X-chromosomal inversion breakpoint disrupting DMD at intron 41. This finding provided molecular confirmation of the clinical diagnosis, which is important as it enables subsequent carrier testing as well as prenatal and preimplantation genetic testing in the family. Further testing of the maternal grandmother indicated that the inversion had likely arisen as a de novo in the patient’s mother, an important finding for appropriate genetic counseling of the family and determining the recurrency risk in future pregnancies.

With the advent of long-read technologies, pathogenic inversions disrupting DMD are being detected and reported more frequently. Considerable variation exists in inversion sizes and breakpoints; however, they all prevent production of the full-length primary muscle dystrophin isoform (Dp427m). Of the previously reported inversions causative for DMD, one additional patient harbored an inversion with a breakpoint located at intron 41 [11], similar to the patient described in the current report. Curiously, despite similar breakpoints within DMD, a neurodevelopmental deficit was present in our patient but not in the patient reported by Chen et al. As neither case involved disruption of additional genes at the inversion breakpoint outside DMD, the different genomic context into which DMD exons 42–79 are relocated may play a role. In particular, this altered context may lead to differences in the expression of DMD isoforms that could be transcribed from the relocated part of the gene, such as Dp140, which has been proposed to be important for neurodevelopment [2]. In our case, the exons 42–79 are relocated to a transcriptionally silent intergenic region within Xp22.2, which may prevent the transcription of DMD isoforms that have been implicated in cognitive and learning impairments. Although other unknown factors may contribute, these findings suggest that in cases of balanced inversions, both rearrangement breakpoints may play an important role in determining the phenotypic differences observed in DMD, warranting further investigations.

Considering genomic technologies, as suggested by Dremsek et al. and supported by this report, OGM is particularly valuable method in clinical scenarios where the analysis can be targeted to reveal SVs in certain disease-causing genes [26]. Although OGM does not provide nucleotide-level breakpoint information or detect SVs < 500 bp, its resolution is sufficient to confirm a clinical diagnosis in defined cases [26]. Mutation-negative DMD cases are a particularly well-suited target group to benefit from OGM analysis, given its monogenic nature and OGM’s ability to detect balanced abnormalities as well as other complex rearrangements that, although rare, are causal for the disease [9, 20–22, 27, 28]. Furthermore, in mutation-negative cases, muscle biopsy is often performed to assess dystrophin expression, with the absence of dystrophin supporting the clinical diagnosis of DMD. Improved genetic analysis could provide a less invasive means of confirming the diagnosis, since the detection of a pathogenic DMD variant would make muscle biopsy unnecessary [29], as in the case of our patient.

In conclusion, our study highlights the utility of OGM as an efficient method for detecting and refining cytogenetic abnormalities at high resolution. Balanced abnormalities, such as inversions, are often among the most difficult SVs to detect with short-read sequencing. However, a growing body of evidence shows that OGM, alongside long-read sequencing techniques, is an efficient method for detecting this SV type and the analysis is not confounded by the repetitive sequences that frequently mediate the formation of inversions. Altogether, the current report underscores the role of pathogenic inversions in DMD patients with negative genetic test results, provides insights into their association with intellectual disability, and supports the use of alternative long-read technologies in mutation-negative cases.

Abbreviations

CK

Creatine kinase

CNV

Copy number variant

DMD

Duchenne muscular dystrophy

FISH

Fluorescence in situ hybridization

indel

Insertion-deletion

MLPA

Multiplex ligation-dependent probe amplification

NGS

Next-generation sequencing

OGM

Optical genome mapping

SNV

Single nucleotide variant

SV

Structural variant

Author contributions

Conceptualization, TM, AH, SSK, PI and AKA; data analysis, TT, LH, TM; original draft preparation, TT, TM, SSK and PI; patient treatment and clinical data, SSK, PI, MT; resources, TM, KP, AH, SSK and AKA; review and editing, all authors. All authors have read and agreed to the published version of the manuscript.

Funding

Open Access funding provided by University of Oulu (including Oulu University Hospital). This work was supported by Helsinki University Hospital Diagnostic Center research fund. TM received funding from research council of Finland (338374, 360442) and Sigrid Jusélius Foundation (220111). SSK received a grant from the Children´s Cancer Foundation Väre and the Finnish Foundation for Pediatric Research.

Data availability

Upon reasonable request.

Declarations

Ethics approval and consent to participate

The research was approved by the HUS Regional Committee on Medical Research Ethics (HUS/2834/2021). Consent to participate has been obtained from all human participants.

Consent for publication

Written consent to publish this paper was obtained from the family.

Competing interests

The authors declare no competing interests.