Large Genetic Study Maps DMD Variant Spectrum in Nearly 1,000 Patients

Researchers at the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University have reported one of the largest single-center analyses of Duchenne and Becker muscular dystrophy genetics to date, characterizing the spectrum of disease-causing variants in 940 unrelated probands tested between March 2022 and May 2025. Using a stepwise workflow that applied multiplex ligation-dependent probe amplification (MLPA) first and exome sequencing (ES) second, the team identified a pathogenic or likely pathogenic DMD variant in 921 of 940 genetically or muscle-biopsy-confirmed cases, a figure corresponding to 97.98 percent of the confirmed cohort.

Duchenne and Becker muscular dystrophies are allelic X-linked recessive neuromuscular disorders caused by pathogenic variants in the DMD gene, which encodes the protein dystrophin. The incidence of DMD/BMD among male newborns in mainland China is approximately one in 4,560, suggesting that nearly 1,000 affected infants are born in the country each year. Although the conditions remain incurable, standardized multidisciplinary management with corticosteroids, rehabilitation, and cardiopulmonary support has been shown to delay disease progression, improve quality of life, and extend survival. Accurate molecular diagnosis is therefore central not only to confirming dystrophinopathy but also to assessing recurrence risk and determining eligibility for mutation-specific or gene-targeted therapies.

The cohort consisted of 924 male and 16 female probands whose ages ranged from birth to 54 years. Participants were recruited on the basis of clinical suspicion of dystrophinopathy, supported by findings such as progressive symmetric muscle weakness, a positive Gowers sign, calf pseudohypertrophy, serum creatine kinase levels greater than five times the upper limit of normal, myopathic electromyography, or muscle-biopsy findings reported as supportive of dystrophinopathy. Patients with alternative molecular diagnoses or definitive diagnoses of other neuromuscular disorders, including spinal muscular atrophy, myasthenia gravis, limb-girdle muscular dystrophy, congenital muscular dystrophy, or Pompe disease, were excluded. Of 961 pedigrees initially collected, 21 were removed from the DMD/BMD analyses because testing revealed pathogenic variants in other neuromuscular disease genes.

The diagnostic strategy reflected the underlying biology of the DMD gene. Because large exon deletions and duplications account for the majority of pathogenic variants, MLPA using the SALSA P034/P035 DMD/Becker kit served as the first-tier test, screening all 79 exons with defined dosage-quotient thresholds and quality controls. Probands with negative MLPA results, or with apparent single-exon deletions that could reflect probe-binding-site variants rather than true deletions, then underwent exome sequencing to detect single-nucleotide variants and small insertions and deletions. Sequencing was performed on an Illumina NovaSeq 6000 platform with a mean depth of at least 100×, at least 98 percent of target bases covered at 20× or more, and capture efficiency of at least 80 percent. All pathogenic and likely pathogenic sequence variants identified by exome sequencing were independently confirmed by PCR and Sanger sequencing before being reported, using the DMD reference transcript NM_004006.4.

The resulting variant spectrum showed that large deletions dominated, accounting for 66.34 percent (611 of 921) of molecularly diagnosed cases, followed by small sequence variants at 20.63 percent (190 of 921), large duplications at 11.73 percent (108 of 921), and complex structural variants at 1.30 percent (12 of 921). Among the deletions, the number of deleted exons ranged from one to all 79. Single-exon deletions were the most common pattern at 22.42 percent of deletion cases, with deletion of exon 45 the most frequent individual event, followed by deletion of exon 51. Deletions spanning exons 45 to 50 were the most frequently observed multi-exon pattern. On the duplication side, single exon 2 duplication was the most common, while duplications involving exons 2 to 20 represented the most frequently affected region, accounting for 34.26 percent of duplication cases.

The 190 small variants comprised 98 nonsense variants (51.58 percent), 47 frameshift variants (24.74 percent), 36 splice-site variants (18.95 percent), and 9 missense variants (4.74 percent). Twenty potentially novel variants, absent from publicly accessible databases and the literature, were also identified, expanding the observed variant spectrum. The authors cautioned, however, that functional RNA validation was not performed for the non-canonical splice-site and deep intronic variants among these, so their predicted effects on splicing should be interpreted cautiously. The 12 complex structural variants included one combined deletion-duplication event, nine cases of multiple discontinuous duplications, and two inversions, one involving an insertion within the chrX:1,474,789–1,721,700 region and the other a large inversion within chrX:1,721,704–31,907,970. These externally reported rearrangements were confirmed at the study center by targeted PCR followed by Sanger sequencing, and all affected patients presented with clinical phenotypes typical of Duchenne muscular dystrophy.

Maternal segregation analysis was performed in 888 families for which samples from both proband and mother were available; in the remaining 52 families the mothers elected not to undergo testing, mainly because of cost or the absence of further reproductive plans. The analysis showed that 279 cases, or 31.42 percent, carried presumed de novo variants, while 609 families, or 68.58 percent, had inherited variants. Germline mosaicism was suspected in 14 families, in which the same variant appeared in more than one offspring despite its absence from maternal peripheral blood. The composition of variant types differed strikingly between the two groups: in the de novo group, large deletions, duplications, and small variants accounted for 80.45 percent, 6.39 percent, and 13.16 percent of cases respectively, whereas in the inherited group the proportions were 59.11 percent, 16.58 percent, and 24.30 percent. Among de novo large deletions, deletions of exons 48 to 50 were the most frequent, at approximately 6.02 percent of cases. The authors emphasized that the de novo classification rested on the absence of the variant in maternal peripheral blood alone, not on testing of multiple maternal tissues or germ cells, so low-level maternal somatic or germline mosaicism cannot be excluded and the presumed de novo proportion should be interpreted with caution. A negative maternal blood result, they noted, does not eliminate recurrence risk in subsequent pregnancies.

Phenotypically, Duchenne muscular dystrophy accounted for 81.00 percent (746 of 921) of the families with identified variants and Becker muscular dystrophy for 19.00 percent (175 of 921). Large deletions predominated in both groups, at 63.40 percent of DMD families and 77.71 percent of BMD families, with BMD deletions occurring predominantly as in-frame variants and deletions of exons 45 to 47 the most frequent, at approximately 14.29 percent. The clustering of deletions within exons 45 to 55, a structurally fragile segment of the gene rich in repetitive sequences and Alu elements prone to non-allelic homologous recombination, was consistent with most global reports and with the TREAT-NMD Global Database, in which large rearrangements account for roughly 80 percent of reported DMD variants. The authors cautioned that direct international comparisons should be interpreted carefully because of differences in cohort composition, referral patterns, classification criteria, and testing strategies, and that multicenter population-based studies are needed to determine whether observed differences reflect population-specific characteristics.

The 16 female probands with heterozygous DMD variants, whose variants included nine exon deletions, two exon duplications, and five small variants, displayed a wide clinical range, from no reported symptoms to elevated creatine kinase levels to clinically manifest dystrophinopathy. Seven of the 16 exhibited mildly elevated creatine kinase. One case, a female with a heterozygous deletion of exons 45 to 50, presented beyond the typical asymptomatic carrier profile, with persistently elevated serum creatine kinase, electromyographic evidence of definitive myopathic changes, and objectively observed gait abnormalities consistent with a Becker-like phenotype. Skewed X-chromosome inactivation is considered the most common cause of symptomatic female carriers, but X-inactivation testing, chromosomal abnormality analysis, and mosaicism assessment were not systematically available in this cohort, so the mechanisms underlying the variable clinical expression could not be established. The authors also noted that the small, heterogeneously referred group of females means the study was not designed to estimate the prevalence or full clinical spectrum of symptomatic carriers.

Nineteen patients with biopsy-supported dystrophinopathy, about 2.02 percent of the cohort, remained molecularly unresolved after both MLPA and exome sequencing. Potential explanations include deep-intronic or regulatory variants, complex structural rearrangements beyond the resolution of these methods, low-level mosaicism, and coverage or alignment limitations of short-read sequencing; the absence of RNA analysis, whole-genome sequencing, and systematic long-read sequencing may also have limited resolution. The authors stressed several sources of bias: this was a single-center, referral-based study rather than population-based screening, so the variant detection rate, mutation spectrum, de novo proportion, and carrier frequency should not be read as population-level estimates, and the provincial distribution of patients, with 42.55 percent from Henan and 57.45 percent from other provinces including Shandong, Shanxi, and Tibet, reflects referral patterns rather than epidemiological prevalence. Nevertheless, the findings refine the contemporary DMD variant spectrum in a Chinese referral population, support the clinical utility of the sequential MLPA-first, ES-second workflow while acknowledging its limits, and provide evidence for molecular diagnosis, inheritance assessment, individualized genetic counseling, and evaluation of eligibility for mutation-specific therapies.

Subject of Research: Genetic diagnosis and variant spectrum of Duchenne and Becker muscular dystrophies in a large Chinese referral cohort

Article Title: Genetic diagnosis and variant spectrum in 940 patients with Duchenne and Becker muscular dystrophies

Article References: Zhou, R., Liu, L., Hua, C., Zhao, Z., Liu, W., & Kong, X. (2026). Genetic diagnosis and variant spectrum in 940 patients with Duchenne and Becker muscular dystrophies. Egyptian Journal of Medical Human Genetics, 27(1), Article 89. https://doi.org/10.1186/s43042-026-00918-5

Image Credits: AI Generated

DOI: 10.1186/s43042-026-00918-5

Keywords: Duchenne muscular dystrophy, Becker muscular dystrophy, DMD gene, MLPA, exome sequencing, genetic diagnosis, de novo variants, germline mosaicism, exon deletions, female carriers, dystrophinopathy, variant spectrum