Recurrent nonsense p.Trp3416* variant in the DMD gene identified in healthy Lebanese individuals: Implications for variant classification and genotype-phenotype correlations

Abstract

Background:

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder caused by pathogenic variants in the DMD gene, leading to dystrophin deficiency and progressive muscle degeneration. Thousands of variants with diverse types have been reported in DMD, contributing to a broad clinical spectrum. While typically associated with severe phenotypes, pathogenic DMD variants may also cause Becker muscular dystrophy (BMD), a milder form with later-onset muscle weakness, or isolated dilated cardiomyopathy with minimal skeletal muscle involvement. Rarely, asymptomatic individuals carry putative pathogenic variants, challenging established genotype-phenotype correlations.

Methods:

This study includes five unrelated Lebanese families who presented for genetic counseling or pre-marital screening and subsequently underwent genetic testing.

Results:

Exome sequencing revealed a predicted protein-truncating variant in exon 71 of DMD p.(Trp3416*) in multiple individuals, including three hemizygous healthy males aged 35, 65 and 67. Despite being classified as pathogenic, the variant's presence in asymptomatic males raises questions about its actual pathogenicity. In silico tools (e.g., Franklin, Varsome, CADD score: 52.0) predicted a strong deleterious effect. The variant is extremely rare in population databases and has conflicting interpretations in ClinVar, previously associated with DMD, BMD, and cardiomyopathy. Although other truncating variants in exon 71 are known to cause DMD, the identification of p.(Trp3416*) in healthy individuals with normal neuromuscular and cardiac function suggests the possibility of alternative splicing, modifier genes, or compensatory mechanisms mitigating the effect of dystrophin loss.

Conclusion:

This study underscores the importance of functional validation and long-term clinical monitoring to refine variant classification and guide accurate genetic counseling.

Introduction

Duchenne Muscular Dystrophy (DMD; OMIM #310200) is a severe X-linked recessive neuromuscular disorder caused by variants in the DMD gene (OMIM *300377), which encodes dystrophin, a crucial structural protein essential for muscle function. ¹ The loss or dysfunction of dystrophin leads to progressive muscle degeneration, with affected individuals typically experiencing delayed motor development, progressive weakness, and loss of ambulation by adolescence. ² In addition to DMD, variants in the DMD gene can also cause Becker Muscular Dystrophy (BMD; OMIM #300376) and DMD-associated dilated cardiomyopathy (DCM; OMIM #302045), both of which result from variations in the severity and location of dystrophin variants. ³

BMD is a milder allelic variant of DMD, characterized by later onset and slower disease progression. Unlike DMD, where dystrophin is nearly absent, individuals with BMD produce a partially functional but truncated dystrophin protein, allowing them to maintain ambulation and mobility for a longer period. Symptoms of BMD include muscle weakness, calf hypertrophy, and cardiac involvement, with significant variability in severity and time course. While some patients lose ambulation in early adulthood, others remain mobile well into later life. ⁴

DMD-associated DCM is another disorder linked to variants in the DMD gene, primarily affecting cardiac muscle rather than skeletal muscle. This condition occurs when dystrophin deficiency leads to progressive cardiac muscle degeneration, resulting in left ventricular dilation, reduced cardiac function, and an increased risk of heart failure. ⁵ Importantly, dystrophin-deficient DCM can manifest in individuals across the full spectrum of skeletal muscle involvement, from those with classic DMD to milder Becker phenotypes, and even in individuals who are clinically asymptomatic with no skeletal muscle weakness.

Collectively, these three disorders, DMD, BMD, and DMD-associated DCM, highlight the critical role of dystrophin in both skeletal and cardiac muscle integrity, with the severity of the disease largely dependent on the extent of dystrophin dysfunction.

While DMD variants are generally associated with severe clinical manifestations, there have been reports of individuals carrying pathogenic variants who remain asymptomatic. For instance, a study by Bai et al. (2022) examined two males carrying the same DMD duplication, one completely asymptomatic and the other with typical features of DMD. Further analysis revealed that the affected individual had a tandem duplication disrupting the DMD open reading frame, whereas the asymptomatic individual carried an extragenic duplication that did not disrupt the coding sequence of DMD. This case underscores the need for accurate variant characterization to properly assess pathogenicity, particularly in the context of genetic counseling. ⁶

In this paper, we report a stop codon variation predicted to be pathogenic in the DMD gene in healthy individuals from five different Lebanese families. This finding underscores the importance of re-evaluating variant classification and highlights the need for functional studies to distinguish true pathogenic variants from benign or low-penetrance variants.

Material and methods

Patients

We herein describe five different unrelated Lebanese families (Figure 1) referred to our clinic for genetic counseling and clinical evaluation.

Figure 1.

Pedigrees of the families included in this study. Probands are indicated by arrows. Individuals carrying the variant are shaded in gray.

Genetic studies

Written informed consents were obtained from the patients and their families for participation and publication.

Isolation of genomic DNA

Written informed consent was obtained from all participants to participate in this study and its publication. EDTA blood samples from all available members of the families were collected for genetic studies. DNA was extracted from leucocytes by standard salt-precipitation methods.

Exome sequencing (ES)

ES was carried out in five individuals indicated by arrows in Figure 1 (Family 1: II-1, Family 2: I-1, Family 3: I-2, Family 4: II-3 and Family 5: II-1). Briefly, the exome was captured and enriched using Agilent SureSelect Human All Exon kit version 5.0 and after adding unique barcodes for each sample, samples were then multiplexed and subjected to sequencing on an Illumina HiSeq 2500 PE100-125. Reads files (FASTQ) were generated from the sequencing platform via the manufacturer's proprietary software. Reads were aligned to the hg19/GRCh37 reference genome using the Burrows-Wheeler Aligner package version 0.7.11. Variant calling was subsequently performed using the Genome Analysis Tool Kit (GATK) version 3.3. Variants were called using high stringency settings and annotated with VarAFT software 1.61 containing information from dbSNP147 and the Genome Aggregation database (gnomAD, http://gnomad.broadinstitute.org). Filtering of the variants was initially performed according to the frequency of the variant in the gnomAD database v2.1.1 (GRCh37) (<0.01% and <50 heterozygous carriers or <5 homo-/hemizygous carriers), and in our in-house database (<1 homozygous occurrence) that includes 980 Lebanese individuals. All remaining variants were assessed by several prediction tools (e.g., SIFT, PolyPhen-2, VariantTaster) and according to the guidelines established by the ACMG (American College of Medical Genetics and Genomics) for variant classification. ⁷ Among the retained variants, (1) those predicted to be benign/likely benign were excluded; (2) those predicted as pathogenic/likely pathogenic by at least one of the prediction tools were evaluated one by one based on the function of the gene and its involvement in Human diseases. As per the ACMG guidelines, secondary findings were reported to the patients during genetic counseling sessions. Following this step, (3) all remaining variants classified as variants of unknown significance in the coding regions and in the −20/+20 intronic boundaries were individually assessed as above, based on the function of the gene and its involvement in Human diseases.

Sanger sequencing

Sanger sequencing was performed in all available members. Genomic sequence of DMD (NM_0004006) was obtained from UCSC Genomic Browser (Feb. 2009 (GRCh37/hg19)). Primers used for PCR amplification were designed using Primer3 software (http://frodo.wi.mit.edu) to amplify the exon 71 of the DMD gene including the candidate variant detected by ES in the patients. PCR reactions were performed using Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA). PCR fragments were run on 1% agarose gel. The fragments were purified using « SIGMA-ALDRICH TM» kit and then sequenced using the Big Dye_Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequence reaction was purified on Sephadex G50 (Amersham Pharmacia Biotech, Foster City, CA), and then loaded into an ABI3500 system after the addition of Hidi formamide. Electropherograms were analyzed using Sequence Analysis Software version 5.2 (Applied Biosystems) and then aligned with the reference sequences using ChromasPro v1.7.6.1 (Technelysium, Queensland, Australia).

Results

Clinical data

Family 1

The pedigree represents a two-generation family where the proband (II-1), a young female who presented for genetic evaluation due to infertility, was found to be a heterozygous carrier of a novel predicted protein-truncating variant in the DMD gene (NM_004006.3: p.(Trp3416*)). Subsequent cascade testing identified the variant at a heterozygous state in two additional heterozygous sisters within the same generation (II-2, and II-3). Parents were tested and the father (I-1), was found to be hemizygous for the variant. All identified carriers are currently asymptomatic. The father (I-1), aged 67 years, underwent thorough clinical and genetic evaluation to assess for potential manifestations of dystrophinopathy. This included a comprehensive physical examination, electrocardiogram (ECG), echocardiography, and serum creatine kinase (CK) level measurement. All findings were within normal limits, and no signs of skeletal muscle or cardiac involvement were observed.

Family 2

The patient was a healthy Lebanese 35-year-old male who presented to our clinic for pre-marital genetic counseling. He had no history of congenital anomalies, developmental delays, neurological disorders, or chronic illnesses. His growth parameters, including height, weight, and head circumference, were within the normal range for his age. Detailed family history revealed no known genetic disorders or evidence of consanguinity. Physical examination was unremarkable, with normal findings in all major organ systems. Standard laboratory tests, including complete blood count, metabolic panel, and urinalysis, were within normal limits. ECG, echocardiography, and serum CK level measurement all revealed normal results. Given the absence of clinical symptoms and the purpose of reproductive risk assessment, ES was performed to screen for potential pathogenic variants associated with autosomal recessive conditions.

Family 3

The female proband (I-2) was referred for genetic evaluation following recurrent miscarriages. Comprehensive clinical and laboratory assessments, including hormonal profiling, pelvic imaging, thrombophilia screening were unremarkable. Karyotype analysis for both partners did not reveal any chromosomal aberration. ES analysis was then performed for the couple.

Family 4

The female proband (II-2) presented for genetic counseling after being identified as a heterozygous carrier of a nonsense variant in the DMD gene during premarital genetic testing, which included ES. She expressed significant concern regarding the risk of transmitting the pathogenic variant to her future children, particularly male offspring who would be at risk of developing the disease. She is the mother of a healthy daughter but is concerned that her daughter may be carrier of the nonsense variant and is planning for additional children. Given the X-linked recessive nature of DMD and the uncertainty of the variant found, she was counseled regarding reproductive options, including prenatal diagnosis and preimplantation genetic testing, as well as the implications for her daughter and other female family members who may require carrier testing in the future. Two years later, the grandparents of the proband presented to our clinic and requested genetic testing. Testing was offered, and it was found that the healthy 65-year-old grandfather (I-1) is a hemizygous carrier of the variant and is asymptomatic. Clinical assessment of the latter including a comprehensive physical examination, ECG, echocardiography, and CK level revealed entirely normal findings.

Family 5

The parents presented to our clinic for genetic counseling concerning their son, a 32-year-old man with a history of intellectual disability, psychomotor delay, and hyperactivity. He experienced two episodes of convulsive seizures during his lifetime. At 17 years old, he was diagnosed with end-stage renal disease and subsequently underwent kidney transplantation. The family history was notable for the patient's sister, who, according to the parents, had the same clinical presentation as her brother but died suddenly at the age of 9. She had a history of severe convulsive seizures that began at the age of 8.

The proband had previously undergone genetic testing through a commercial laboratory, which reported a nonsense variant in the DMD gene classified as likely_pathogenic. However, upon evaluation of the clinical presentation and family history, our team found that the phenotype was not consistent with dystrophinopathy.

Genetic analysis

ES data of each of the tested individuals was analyzed in accordance with the specific symptoms and medical history provided during the referral process. Surprisingly, this analysis identified a likely pathogenic variant: p.(Trp3416*) in exon 71 of the DMD gene (NM_004006.2) (chrX(hg38):NC_000023.11:g.31177947C > T; c.10247G > A), in the five tested patients. This variant is predicted to create a premature translational stop signal and was identified in all five families.

This variant is absent from our in-house database of 980 individuals but has been identified six times in gnomAD among a total of 1,206,155 alleles (rs201217593, allele frequency: 0.000004974, accessed on February 21, 2025), with only one occurrence at hemizygous state. This variant is predicted to be pathogenic by both Franklin (https://franklin.genoox.com/clinical-db/variant/snp/chrX-31196064-C-T) and Varsome ⁸ and is classified as a loss-of-function variant with a CADD PHRED score of 52.0 (https://cadd.gs.washington.edu/). This variant was also found to result in a nonsense variant in multiple DMD transcripts that include this exon, resulting in predicted truncations at different positions depending on the transcript isoform (NM_000104.4:c.10223G > A; p.W3408*, NM_004009.3:c.10235G > A; p.W3412*, NM_004010.3:c.9878G > A; p.W3293*, NM_004011.4:c.6224G > A; p.W2075*, NM_004012.4:c.6215G > A; p.W2072*, NM_004013.4:c.2867G > A; p.W956*, NM_004014.4:c.2060G > A; p.W687* and NM_004015.4:c.1043G > A; p.W348*). Given that all these isoforms share the same open reading frame in this region, the predicted truncation is consistent across them.

The detected variant has been previously reported once in the literature as a putative loss-of-function variant associated with a negative phenotype. ⁹ However, ClinVar lists conflicting classifications of pathogenicity for this variant (Variation ID: 374132): Pathogenic (3), Likely Pathogenic (4), and Uncertain Significance (4). Indeed, ClinVar data for the DMD p.(Trp3416*) variant shows conflicting classifications of pathogenicity across different submissions. It has been reported as pathogenic in association with DMD (RCV001035227.7, Dec 6, 2023), dilated cardiomyopathy 3B, and BMD (RCV000763208.3, Oct 31, 2018), as well as a general cardiovascular phenotype (RCV002379278.4, Aug 8, 2024). Additionally, it has been classified as likely pathogenic for BMD (RCV003470367.2, Nov 21, 2023; RCV001420737.4, Aug 7, 2023) and for a phenotype including cardiomyopathy, clinodactyly of the 5^th finger, pectus excavatum, proptosis, and ptosis (RCV000415019.3, Oct 3, 2014). However, one submission (RCV002248648.2, May 4, 2022) classified it as variant of uncertain significance for a general cardiovascular phenotype. Notably, a more recent submission (RCV001764346.12, Jul 16, 2024) reports conflicting classifications of pathogenicity, highlighting the ongoing uncertainty regarding the variant's clinical impact. This variant has been recently submitted by our team to ClinVar (Accession: SUB15436700; under evalutaion) to update the findings.

Discussion

We report in this paper the identification of a nonsense variant in the DMD gene (p.Trp3416*) in five different Lebanese families who presented with different clinical scenarios seeking genetic counseling. The identification of this nonsense variant, classified as pathogenic in multiple databases, create considerable stress, particularly for pregnant carrier women who consider their babies to be at high risk of developing DMD. Notably, three hemizygous individuals (Family 1: I-1, Family 2: I-1and Family 4: I-1), aged 67, 35 and 65 years respectively, exhibited no clinical signs of any DMD-related disorders which provided reassurance and facilitated the genetic counseling process. These findings are in line with the observations reported by Torella et al., ¹⁰ suggesting that a nonsense mutation in DMD should not necessarily be classified automatically as a null allele, and this consideration is crucial in the context of genetic counseling.

Since this variant was identified in different unrelated families of diverse Lebanese origin, haplotype analysis based on ES data was performed manually across the different families and revealed that no common haplotype is shared among them. This finding argues against a recent common ancestor and suggests that the variant may have arisen independently or represents an older, more widely distributed allele in the population.

This identified nonsense variant is predicted to introduce a premature stop codon, shortening the protein length from 3684 amino acids to 3416. It is expected to result in a truncated or absent dystrophin protein in all transcripts, consistent with known loss-of-function mechanisms underlying DMD-related disorders. Given the well-established pathogenicity of loss-of-function variants in DMD, this variant is in theory a strong candidate for being disease-causing in affected individuals. Moreover, the rarity of p.(Trp3416*) in population databases further supports its pathogenic potential. Its extremely low allele frequency in gnomAD aligns with expectations for pathogenic variants in X-linked recessive disorders such as DMD and BMD. Additionally, the absence of this variant in our in-house underscores its rarity and potential disease association.

Furthermore, in-silico predictive evidence supports its pathogenicity. Indeed, computational tools such as Franklin and Varsome classify this variant as pathogenic, and its exceptionally high CADD Phred score of 52.0 suggests a severe deleterious effect. Moreover, different truncating variants within exon 71, have been previously linked to milder BMD phenotypes.^11,12 This is in line with the fact that certain truncating variants in this region may allow for partial retention of dystrophin function, resulting in a spectrum of DMD-disease severity. ¹⁰

Despite this compelling evidence, conflicting classifications in ClinVar highlight the ongoing uncertainty surrounding this variant's clinical impact. While multiple ClinVar submissions categorize p.(Trp3416*) as pathogenic or likely pathogenic for DMD and BMD, some submissions report uncertain significance, particularly in relation to broader cardiovascular phenotypes. This discrepancy may come from the fact that clinical geneticists often rely on computational protein predictions to assess the potential impact of genetic variants, rather than conducting functional in vitro analyses. While these predictive tools can provide useful insights, they do not fully capture the complexity of how a variant might affect gene function in a living organism. On the other hand, in vitro analyses are costly and time-consuming and cannot be performed for every newly identified variant. As a result, without experimental validation through in vitro studies, genetic diagnoses can be challenging, as the predictions may not always accurately reflect the actual biological consequences of the variant.

Given these findings, additional functional studies and comprehensive clinical assessments of individuals harboring this variant are needed specifically that the latter is close to the 3′ end of the ORF. RNA-based assays or protein expression studies could help determine whether the truncation results in a complete loss of dystrophin or if some residual function is retained. However, since the individuals carrying this variant are asymptomatic, a complete loss of dystrophin is unlikely. Instead, the variant might cause a partial defect that could be quantitative, reducing overall dystrophin levels, or qualitative, altering the protein's structure or function. Of note, some algorithms developed to predict the effect of sequence changes on RNA splicing suggest that this variant may disrupt the consensus splice site: SPiP ¹³ predicts a 54% risk of Exonic Splicing Enhancer (ESE) disruption, whereas SpliceAI ¹⁴ predicts no splicing consequence; highlighting a lack of concordance among splicing prediction tools. However, as previous studies have shown that exon 71 undergoes significant alternative splicing, the likelihood of exon skipping is high. ¹⁵ This could mitigate the functional impact of the variant by resulting in an in-frame deletion rather than a truncating mutation, potentially explaining the absence of clinical symptoms in affected individuals. In this particular case, obtaining muscle biopsy samples for such analyses is challenging, especially from asymptomatic or healthy individuals, which limits the ability to directly assess dystrophin expression and function in these cases. Nevertheless, an alternative approach could involve the generation of myogenic cell lines from urine-derived induced pluripotent stem cells, which may serve as a non-invasive model system for future studies.

In our case, five Lebanese families that were well-followed and thoroughly investigated do not present any clinical signs of DMD-related spectrum of diseases and have normal serum CK levels. Although few male carriers of DMD variants (particularly in-frame variants or variants with low-level dystrophin expression) have been reported as asymptomatic or presenting very mild symptoms in late adulthood, these typically exhibit high CK levels ¹⁶ which are not observed in the hemizygous cases currently reported. This suggests that the likelihood of late-onset manifestations in the studied cases is extremely low. This observation, along with all the points discussed above, raise several questions: Do other genetic factors in the Lebanese population compensate for the effect of this variant, rendering it benign, while in other populations, the absence of these components makes it deleterious? Alternatively, should we reclassify this variant as likely benign? Given that recent research has also shown that some DMD duplications, previously thought to be pathogenic, have been reclassified as benign following further genetic analysis; cautious interpretation of DMD variants in clinical settings is indeed required. ¹⁷

Altogether, these observations highlight the challenges encountered in genetic counseling of these families. Indeed, while the detected variant in DMD is classified by several prediction tools as likely pathogenic, yet familial segregation data and clinical observations suggest a benign or non-penetrant effect. This discordance can create significant uncertainty for both clinicians and patients. In such cases, the counselor must carefully balance the available public database classifications with actual evidence from the family, including asymptomatic hemizygous early middle-aged and old-adults males and absence of phenotypic features typically associated with the variant. This challenge is further compounded by the emotional burden it places on families who are trying to make informed reproductive or medical decisions in the face of conflicting information. In these scenarios, integrating detailed family studies, functional data when possible, and longitudinal follow-up becomes essential to providing accurate risk assessment and minimizing unnecessary anxiety or interventions.

In conclusion, while the available evidence strongly suggests that the p.(Trp3416*) variant in DMD is pathogenic, particularly in the context of DMD-related disorders, ongoing discrepancies in ClinVar highlight the need for further validation. Future studies integrating functional characterization, patient phenotyping, and long-term follow-up will be essential to refine its clinical classification and improve genetic counseling for affected individuals and their families. Importantly, public data sharing through variant databases such as ClinVar and LOVD is crucial to ensure collective progress in variant interpretation. A coordinated effort from all laboratories to share genotype -phenotype data will help build reliable, evidence-based classifications and ultimately improve patient care.