Rethinking the pathogenicity of intragenicDMDduplications detected by carrier screening: high prevalence of non-tandem duplications revealed by long-read sequencing

Rethinking the pathogenicity of intragenic DMD duplications detected by carrier screening: high prevalence of non-tandem duplications revealed by long-read sequencing | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Rethinking the pathogenicity of intragenic DMD duplications detected by carrier screening: high prevalence of non-tandem duplications revealed by long-read sequencing Qiliang Ding , Jagadheshwar Balan , Noemi Vidal-Folch , Angela M. Pickart , View ORCID Profile Guangchao Sun , Jesse R. Walsh , Ramanath Majumdar , Eric W. Klee , Stephen J. Murphy , View ORCID Profile Devin Oglesbee , Ross A. Rowsey , Linda Hasadsri doi: https://doi.org/10.1101/2025.04.10.25325596 Qiliang Ding 1 Division of Laboratory Genetics and Genomics, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: ding.qiliang{at}mayo.edu hasadsri.linda{at}mayo.edu Jagadheshwar Balan 2 Department of Quantitative Health Sciences, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Noemi Vidal-Folch 1 Division of Laboratory Genetics and Genomics, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Angela M. Pickart 1 Division of Laboratory Genetics and Genomics, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guangchao Sun 2 Department of Quantitative Health Sciences, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Guangchao Sun Jesse R. Walsh 2 Department of Quantitative Health Sciences, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ramanath Majumdar 3 Advanced Diagnostics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eric W. Klee 2 Department of Quantitative Health Sciences, Mayo Clinic , Rochester MN, 55905, USA 4 Center for Individualized Medicine, Mayo Clinic , Rochester MN, 55905, USA 5 Department of Clinical Genomics, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen J. Murphy 3 Advanced Diagnostics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Devin Oglesbee 1 Division of Laboratory Genetics and Genomics, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Devin Oglesbee Ross A. Rowsey 1 Division of Laboratory Genetics and Genomics, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Linda Hasadsri 1 Division of Laboratory Genetics and Genomics, Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester MN, 55905, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: ding.qiliang{at}mayo.edu hasadsri.linda{at}mayo.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Purpose The pathogenicity of intragenic duplications depends on their structural configuration. Tandem duplications often disrupt reading frames and cause gene loss-of-function, whereas interspersed (non-tandem) duplications are largely benign. When the configuration cannot be determined, current guidelines presume a tandem structure, leading to some laboratories automatically classifying such variants as likely pathogenic or pathogenic. This study evaluates the validity of this presumption for DMD , in patients with and without clinical indications of dystrophinopathy. Methods We performed high-coverage whole-genome long-read sequencing on 15 patients with intragenic DMD duplications. Four patients had clinically indicated dystrophinopathy testing, while in the remaining 11 patients, the duplications were detected without clear indications of dystrophinopathy (e.g., “incidentally detected” through carrier screening). Results All four patients with clinical indications had tandem duplications. In contrast, 64% (7/11) of the incidentally detected cases had interspersed duplications, with four subsequently re-classified as likely benign, two likely pathogenic, and one uncertain. These duplications were often complex, involving co-duplications or co-deletions with other regions. Conclusion Our findings challenge the presumption that intragenic DMD duplications are predominantly tandem. This highlights the need for a cautious variant interpretation approach, particularly in carrier screening and other settings where variants are identified without indications of dystrophinopathy. Introduction The DMD gene is associated with X-linked dystrophinopathies (also referred to as DMD -related disorders), including Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD -associated dilated cardiomyopathy. Patients with DMD who are assigned male at birth (AMAB) typically exhibit muscle weakness in early childhood, loss of ambulation by age 10–12, and death by age 30. Compared with DMD, BMD is a milder disease characterized by later onset, slower progression, and longer life expectancy. 1 DMD and BMD are relatively common genetic conditions, with a prevalence of 1.38 per 10,000 U.S. AMABs aged 5–24 years. 2 An Israeli study estimated the carrier frequency of dystrophinopathy in individuals assigned female at birth (AFAB) to be 1 in 1,374. 3 Due to the carrier frequency and significant disease severity, the American College of Medical Genetics and Genomics (ACMG) recommends offering DMD carrier screening to all patients who are pregnant or considering pregnancy. 4 Intragenic duplications—duplications involving one or more exons but fully contained within a given gene— are a significant source of DMD pathogenic variants, with studies attributing 5–20% of dystrophinopathy cases to these variants. 1 , 5 Nonetheless, not all intragenic DMD duplications are pathogenic. Intragenic duplications can be either tandem or interspersed (also known as non-tandem or insertional). In tandem duplications, the duplicated segment is joined adjacent to the original segment in the same orientation, therefore may disrupt the reading frame and lead to pathogenicity. In contrast, interspersed duplications generally do not disrupt the reading frame and are possibly benign. The disease-causing intragenic DMD duplications found in dystrophinopathy patients are in tandem. 6 However, the structural configuration (i.e., tandem vs. interspersed) of such variants is not routinely assessed in clinical laboratory practice, partly due to technical limitations. Most current clinical assays for copy-number detection are unable to distinguish between tandem and interspersed duplications. These assays include multiplex ligation-dependent probe amplification (MLPA), chromosomal microarray analysis (CMA), and most next-generation sequencing (NGS) assays. Instead, current variant interpretation guidelines—namely, the application of PVS1 7 and PM4 8 —presume intragenic duplications are in tandem unless proven otherwise (hereafter referred to as the “tandem presumption”). While the tandem presumption is likely accurate in patients with a strong clinical indication for dystrophinopathy testing (i.e., personal or family history), little is known about intragenic DMD duplications detected in individuals without such an indication (hereafter referred to as “incidentally detected”). This represents a critical knowledge gap, as many patients undergo DMD genetic testing as part of carrier screening in the absence of a clinical indication of dystrophinopathy. If the tandem presumption is inaccurate in these patients, it could lead to overestimation of variant pathogenicity—potentially resulting in unnecessary invasive procedures for diagnostic fetal testing and/or even pregnancy termination. In this study, we performed Oxford Nanopore Technologies (ONT)-based long-read sequencing on samples from 15 patients with intragenic DMD duplications. The duplications were in tandem in all four cases with a clinical indication of dystrophinopathy. However, among the 11 incidentally detected cases, seven (64%) involved interspersed duplications. We also developed DMDuper, a computational tool that accurately distinguishes tandem from interspersed intragenic DMD duplications using long-read sequencing data, with 100% accuracy across the 15 cases. Taken together, our findings challenge the tandem presumption for incidentally detected intragenic DMD duplications (e.g., those identified by carrier screening) and highlight the need for a cautious approach to variant classification in these cases. Materials and Methods DNA was extracted using Qiagen (Hilden, Germany) or Chemagic (Revvity, Waltham, MA) kits, which was then processed with the Short Read Eliminator kit (PacBio, Menlo Park, CA), and subsequently sheared to 30 kb on Megaruptor 3 (Seraing, Belgium). Library was prepared using the SQK-LSK114 kit and sequenced using R10.4.1 flow cells on a PromethION 24 (ONT, Oxford, UK) for 72 h, with one sample per flow cell. Base calling was performed by Dorado version 0.8.0 ( https://github.com/nanoporetech/dorado ). Alignment to GRCh38 was performed using Winnowmap2 version 2.03 ( https://github.com/marbl/Winnowmap ). 9 The Integrative Genomics Viewer (IGV) was used for manual review of the BAM files. Long-range phasing was manually performed using IGV by identifying reads that share the same single-nucleotide variants. The UCSC BLAT tool was used to investigate read clips ( https://genome.ucsc.edu/cgi-bin/hgBlat ). All genomic coordinates in this manuscript are in GRCh38/hg38. In addition, we developed DMDuper, a bioinformatics tool that can automatically distinguish between tandem and interspersed duplications. Briefly, DMDuper first recognizes a DMD duplication by detecting soft-clipped read signatures at the breakpoints. DMDuper then extracts clipped reads at the breakpoints and performs haplotype-aware assembly using Flye version 2.9.4 ( https://github.com/mikolmogorov/Flye ). The assembled contigs are mapped to the reference genome using minimap2 version 2.18 10 ( https://github.com/lh3/minimap2 ), and the alignments are used to infer the configuration. DMDuper is available at https://github.com/jagadhesh89/dmduper . Results We analyzed 15 samples with intragenic DMD duplications using ONT high-coverage whole-genome long-read sequencing (see Table S1 for sequencing metrics). Four of the 15 cases had a personal or family history consistent with dystrophinopathy (DMD-1 to DMD-4, Table 1 ), while the remaining 11 did not have a clear indication of dystrophinopathy (DMD-5 to DMD-15, Table 1 ). These 11 cases included five ascertained through carrier screening and six that underwent microarray testing for other indications (e.g., congenital heart disease, short stature, autism). View this table: View inline View popup Download powerpoint Table 1. Clinical and laboratory information of the cases included in this study. In all four cases with a clinical indication of dystrophinopathy (DMD-1 to DMD-4), the duplications were confirmed to be in tandem ( Figures 1A–D ). This finding aligned with expectations, as pathogenic intragenic DMD duplications are known to occur in tandem. 6 Download figure Open in new tab Figure 1. Eight cases of tandem intragenic DMD duplications. The blue-colored regions denote duplications. The arrows indicate orientation of the genomic segments. (A–D) Cases with a personal or family history of dystrophinopathy. (E–H) Cases with incidentally detected duplications. Strikingly, only four of the 11 cases with incidentally detected intragenic DMD duplications were discovered to be in tandem (DMD-5 to DMD-8; Figures 1E–H ). For three of the four cases, the duplicated exon contents had been previously reported in dystrophinopathy patients. 11 , 12 The fourth case (DMD-8) harbored a duplication of exons 31–40, which had not been described before. Given that these duplications were confirmed to be in tandem, they are consistent with carrier status for dystrophinopathy. The remaining seven (64%) of the 11 cases (DMD-9 to DMD-15) were found to harbor interspersed duplications. The duplicated exon content varied across the cases ( Table 1 ), which suggests that they arose from distinct mutagenic events. Interestingly, four cases (DMD-9 to DMD-12) with interspersed duplications had “co-duplication” of DMD exons with another locus ( Figure 2 ). The co-duplicated locus varied among the four cases but was consistently on the short arm of the X chromosome, 1.7–30.8 Mb from the duplicated DMD exons. In these cases, split reads and read depth supported the presence of a “co-duplication cassette” containing the duplicated DMD exons fused with the co-duplicated locus, in the same orientation. However, split reads alone could not resolve whether this cassette was inserted within the DMD gene or into the region where the co-duplicated locus originated. These two possibilities may be differentiated using long-range phasing across the cassette (illustrated in Figure 2A ). Download figure Open in new tab Figure 2. Four cases of interspersed intragenic DMD duplications characterized by co-duplication events. The blue-colored regions denote duplications. The arrows indicate orientation of the genomic segments. (A) Schematic overview of the co-duplication events, with two possible structural configurations of the co-duplication cassette, supported by split reads (purple and green) and copy number profiles. (B–E) Detailed co-duplication patterns in DMD-9 to DMD-12. Each case involves the duplication of specific DMD exons (region 5 in panels B–D and region 2 in panel E) alongside a region from the short arm of the X chromosome, located at varying distances from DMD (region 2 in panels B–D and region 5 in panel E). Long-range phasing using SNVs resolved the structural configuration in DMD-9 to DMD-11 (panels B–D), while the configuration remained unresolved in DMD-12 due to insufficient SNV data (panel E). DMD-9 involved the co-duplication of an 88-kb region containing DMD exons 49–50 along with a 135-kb region originating 3.4 Mb telomeric to the DMD duplication harboring no RefSeq genes. DMD-10 involved the co-duplication of a 284-kb region containing DMD exons 50–55 along with a 295-kb region originating 9.1 Mb telomeric to the DMD duplication containing part of PHEX (exon 12 to the 3’-end) and the full CBLL2 gene, both of which are located on the opposite strand relative to DMD. PHEX is associated with X-linked dominant hypophosphatemic rickets, while CBLL2 is not currently associated with any diseases. In both cases, long-range phasing using single-nucleotide variants (SNVs) clarified that the co-duplication cassettes inserted into the regions where the co-duplicated loci originated (i.e., an intergenic region in DMD-9, and adjacent to PHEX and CBLL2 in DMD-10; Figures 2B , 2C, S1, S2), and did not disrupt the reading frame of any genes. Therefore, we concluded that the intragenic DMD duplications in DMD-9 and DMD-10 were likely benign. DMD-11 involved the co-duplication of a 77-kb region containing DMD exons 63–67 along with a 47-kb region originated 30.8 Mb telomeric to the DMD duplication and includes part of PPP2R3B (5’-start to exon 8). PPP2R3B is not currently associated with any diseases. In contrast to DMD-9 and DMD-10, long-range phasing revealed that the co-duplication cassette had inserted within DMD ( Figures 2D and S3). Thus, the duplicated DMD and PPP2R3B (both on the reverse strand) exons may affect the reading frame of DMD , likely indicative of true carrier status for dystrophinopathy, although the exact impact on the DMD reading frame is uncertain due to the inclusion of exons from PPP2R3B . DMD-12 was found to have co-duplication of a 260-kb region containing DMD exons 52–57 along with a 108-kb region originating 1.7 Mb centromeric to the DMD duplication containing no RefSeq genes. Due to the paucity of SNVs, long-range phasing was not possible for this sample. Therefore, the insertion location of the co-duplication cassette could not be determined, and this duplication remains classified as a variant of uncertain significance (VUS; Figure 2E ). In DMD-13, previous CMA testing detected duplications of DMD exons 3–9 and exons 45–48 (region 6 and 2, respectively, in Figure 3A ). Long-read sequencing additionally identified a 27-bp intronic duplication (region 4 in Figure 3A ). Split-read analysis revealed that the duplication cassette included regions 2 and 4 in an inverted orientation, fused with region 6 in the direct orientation, and that the cassette had inserted within the DMD gene ( Figures 3A and S4). The net effect of this variant is the duplication of exons 3–9 (region 6) in the DMD reading frame, which had been previously reported in DMD patients. 13 Therefore, this variant was classified as likely pathogenic, consistent with carrier status for a DMD -related disorder. Download figure Open in new tab Figure 3. Three additional cases of interspersed intragenic DMD duplications. The blue- and red-colored regions denote duplications and deletions, respectively. The arrows indicate orientation of the genomic segments. (A) A complex variant involving the duplication of three distinct segments within DMD . (B) Duplicated DMD exons 5–7 inserted 87.2 Mb away on the long arm of the X chromosome, in an inverted orientation, at the site of a 109-kb deletion. (C) Duplicated DMD exons 3–4 inserted into the repetitive 9q12 (9qh) region. In DMD-14, long-read sequencing revealed that the duplication of DMD exons 5–7 had inserted about 87.2 Mb away from their original location in an inverted orientation. At the site of the insertion, there was also a 109-kb deletion that included RHOXF2B and RHOXF1 , two genes with no known disease association ( Figure 3B ). Because this variant did not affect the DMD reading frame, it was classified as likely benign. In DMD-15, long-read sequencing revealed that the duplicated DMD segment containing exons 3–4 was flanked on both sides by repetitive sequences from 9q12 (i.e., the 9qh region; Figure 3C ). Based on long-range phasing, we concluded that the duplicated DMD exons 3–4 had inserted into 9q12. Therefore, this variant was also classified as likely benign. We also evaluated the performance of DMDuper in automatically determining the structural configuration of the duplications (i.e., tandem vs. interspersed). DMDuper achieved 100% concordance with the results from manual review (examples in Figure S5). Discussion Clinical Significance of Our Findings Intragenic duplications of DMD have been extensively studied, but most of these studies have focused on patients affected by dystrophinopathy. Few, if any, systematic studies have specifically addressed patients with “incidentally detected” (i.e., asymptomatic individuals with no known family history) intragenic DMD duplications, despite the increasing relevance of this issue in pre-conception and prenatal genetic counseling. Our study is the largest to date to examine this group of patients. The study design aimed to minimize ascertainment bias in the group of incidentally detected duplications by selecting samples based solely on specimen availability (5 µg of high-molecular-weight DNA), without any additional criteria. Previous reports have noted that most (~80%) of duplications throughout the genome are tandem and in direct orientation 14 . Strikingly, we found that 64 percent (7/11) of cases in our incidental detection cohort had interspersed duplications, of which four were subsequently classified as likely benign, two were classified as likely pathogenic, and one remained VUS due to the inability to fully resolve its structural configuration. Notably, had the current guidelines on the tandem presumption been followed, all of these variants would have been classified as pathogenic or likely pathogenic. Thus, our findings underscore the necessity of a more cautious and nuanced approach when interpreting intragenic DMD duplications detected without a clinical indication of dystrophinopathy. Previous case reports have detected interspersed intragenic DMD duplications and likewise note the complexities of interpreting such variants in this gene. Bai et al. 15 reported a male individual with DMD exons 56–61 duplication who was asymptomatic. PacBio long-read sequencing revealed that the duplication was interspersed, inserted over 4 Mb away from its original location. Two recent conference abstracts 16 , 17 described four cases of interspersed duplications involving DMD exons 45–51 inserted into 17q21.1, in patients without a clinical indication of dystrophinopathy. It is important to note that both exons 45–51 and 56–61 duplications have been reported in dystrophinopathy patients (presumably in tandem), suggesting that exon content alone cannot reliably distinguish between tandem and interspersed duplications. Our study, with a larger sample size, supports the substantial prevalence of interspersed configurations among incidentally detected intragenic DMD duplications. The Advantages of Long-read Sequencing for DMD Duplication Analysis An important methodological question is whether short-read next-generation sequencing (SR-NGS), when it covers all exons and introns of DMD , is sufficient to resolve the structural configuration of intragenic DMD duplications, or if long-read sequencing is necessary. We argue that long-read sequencing is highly advantageous over SR-NGS. Firstly, many of the interspersed duplications identified in this study have breakpoints within repetitive regions defined by RepeatMasker, which would be difficult to align and analyze accurately with short reads. This limitation of SR-NGS is best exemplified by DMD-9. This patient was initially identified as positive for an intragenic DMD duplication by carrier screening, and subsequently underwent a targeted SR-NGS clinical test at an external laboratory using a custom-designed DMD capture probeset. Despite capturing all DMD exons and introns (including the breakpoint regions identified by our study), SR-NGS was unable to resolve the duplication breakpoints in DMD-9, and the variant was interpreted as pathogenic by the external laboratory. This result had prompted chorionic villus sampling to determine the genotype in the patient’s male fetus—fortunately, the fetus did not carry the duplication. Using long-read sequencing, we were able to reveal that the duplicated exons in DMD-9 were inserted into an intergenic region outside DMD , suggesting that the variant is in fact likely benign ( Figure 2B ), and the invasive prenatal testing may not have been indicated. In addition, long-read sequencing enabled the identification of a co-duplication cassette in several cases, where DMD exons were duplicated alongside another locus (see Results). If the co-duplication cassette had inserted into the region where the co-duplicated locus originated (as in DMD-9 and DMD-10), it is unlikely to affect the DMD reading frame. However, if the cassette had inserted into the DMD gene itself (as in DMD-11), it may disrupt the DMD reading frame and be likely pathogenic. Long-range phasing across the co-duplication cassette is necessary to distinguish between these two scenarios, which is possible with long-read sequencing but not with SR-NGS. Towards a More Cautious Variant Interpretation Approach This study has important implications for variant interpretation of intragenic DMD duplications detected in patients with no known personal nor family history of dystrophinopathy, including those undergoing carrier screening. For variants detected by an assay that cannot resolve structural configuration (i.e., “dosage-only”), such as MLPA, CMA, and most NGS assays, the laboratory should reconsider the presumption that intragenic duplications are in tandem. This presumption is inaccurate in 64% of the incidentally detected intragenic DMD duplications we examined. Without further information, we believe it is most prudent to classify incidentally detected intragenic DMD duplications as VUS until proven otherwise. We also recommend considering long-read sequencing (or less optimally, a SR-NGS assay that sequences all exons and introns in DMD ), when available, to be used to clarify the structural configuration of the duplication. Cytogenetic assays, e.g., fluorescence in situ hybridization (FISH), may assist in clarifying the location of the duplication. 16 If the duplication was found to be in tandem, PVS1 (out-of-frame) 7 , or PVS1 with modified strength or PM4 (in-frame) 8 may be applied. On the other hand, if the duplication was found to be interspersed, it is important to assess the impact on the DMD reading frame. If the duplication does not impact the reading frame of DMD or any other disease-causing genes, it may be classified as likely benign (as in DMD-9 and DMD-10). Otherwise, it may be classified as likely pathogenic (as in DMD-11). In cases where clarification of the structural configuration is impossible or inconclusive, it is crucial to carefully consider family history and perform familial variant testing to help interpret the pathogenicity of the variant. For example, detection of the variant in carefully phenotyped adult asymptomatic male relatives would support a likely benign classification. We summarized the proposed variant classification approach in Figure 4 . Download figure Open in new tab Figure 4. Proposed decision tree for interpretation of intragenic DMD duplications. This flowchart outlines the recommended steps for interpreting intragenic DMD duplications, emphasizing that they should not be presumed to be tandem in the absence of a clinical indication of dystrophinopathy. If additional tests to clarify the structural configuration are inconclusive or unavailable, and familial analysis is uninformative, these variants should be classified as variants of uncertain significance (VUS). Limitations and Future Directions This study represents the largest investigation of the structural configurations of incidentally detected DMD duplications to date. However, the sample size of 15 cases is still relatively modest, and larger studies are needed to further confirm these findings. Despite the sample size, the detection of a substantial number of interspersed intragenic DMD duplications supports the hypothesis that these variants are not rare. We emphasize that this issue should be brought to the attention of clinical laboratory geneticists, maternalfetal medicine specialists, and prenatal genetic counselors. Ethics Declaration This study was conducted as a Quality Improvement initiative at the Department of Laboratory Medicine and Pathology, Mayo Clinic, to improve interpretation of DMD genetic testing results. The Mayo Clinic Institutional Review Board (IRB) determined that no IRB approval was required for this project. Conflict of Interest This study was conducted under a joint development collaboration agreement between Mayo Clinic and Oxford Nanopore Technologies (ONT). The authors declare no conflicts of interest. Data Availability The data generated by this study is available from the corresponding authors upon request. Acknowledgements The authors thank the staff in the Department of Laboratory Medicine and Pathology, Mayo Clinic, for preparing samples and generating data used in this study. 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