The biology behind spinal muscular atrophy explains why this region has long frustrated diagnosticians. The disease is caused by loss or mutation of the SMN1 gene, which encodes a protein essential for the survival of motor neurons. Humans, however, carry a nearly identical backup copy called SMN2, distinguished by a single nucleotide change at position c.840 in exon 7. That one base difference alters how SMN2 pre-messenger RNA is spliced, so most SMN2 transcripts are translated into a truncated, unstable protein that is rapidly degraded. Each SMN2 copy produces only about ten percent of the functional protein generated by SMN1, and the proportion varies across tissues. Because the two genes are more than ninety-nine percent identical, distinguishing them requires reading across the entire roughly 28.5-kilobase locus, something short-read sequencing struggles to accomplish reliably.

Traditional testing reflects these limitations. Quantitative PCR can detect homozygous deletions of SMN1 exons 7 and 8, the molecular signature of most SMA cases, but it cannot report precise copy numbers or identify point mutations. Multiplex ligation-dependent probe amplification, or MLPA, reliably measures SMN1 and SMN2 copy numbers and detects exon rearrangements, yet it cannot resolve haplotypes or sequence-level variants. Neither method can determine whether a person with two SMN1 copies carries both on one chromosome or one on each, a distinction that matters enormously for reproductive risk. A so-called 2+0 silent carrier, with both copies on a single chromosome, appears normal on standard screening but faces the same one-in-four chance of having an affected child as a classic 1+1 carrier when paired with another carrier.

Single-molecule real-time sequencing sidesteps these obstacles through sheer read length. In the new study, researchers at Zhangzhou Municipal Hospital extracted genomic DNA from peripheral blood, amplified the full-length SMN1 and SMN2 genes together with their downstream regions using long-range PCR, and ligated the amplicons into dumbbell-shaped SMRTbell libraries. These libraries were sequenced on a Sequel II CNDx platform in circular consensus sequencing mode for thirty hours, generating high-fidelity reads that span the entire locus. Because each read covers the whole gene, the c.840 site unambiguously labels each molecule as SMN1 or SMN2, allowing the software to count copies of each gene separately and scan for point mutations at the same time. The technology carries no GC bias and handles the highly repetitive sequence architecture of this region that defeats shorter reads.

The clinical validation was striking. Among the 771 screened individuals, SMRT sequencing and qPCR produced fully concordant results in one hundred percent of samples. The most common genotype was two copies each of SMN1 and SMN2, found in 55.25 percent of participants, followed by a 2:1 ratio in 29.44 percent. Two SMN1 copies were present in 91.18 percent of the cohort, but the distribution revealed important outliers: 2.33 percent of individuals carried only one SMN1 copy and were classified as deletion carriers, yielding a carrier frequency of 2.33 percent, consistent with previously reported rates of 1.2 to 2.2 percent in Chinese populations. More provocatively, 6.36 percent carried three SMN1 copies and one individual carried four, indicating that at least two SMN1 genes sit on a single chromosome in these people, a configuration that standard qPCR cannot distinguish from the benign two-copies-on-two-chromosomes arrangement.

That finding carries direct reproductive consequences. Individuals with elevated SMN1 copy numbers may be silent carriers whose risk is invisible to conventional screening, since qPCR reports only total copy number without allele distribution. The study authors note that definitive assignment of 2+0 status still requires family-based haplotype phasing and pedigree analysis, which lay beyond the scope of the current work. Even so, the results underscore a hidden reservoir of reproductive risk in the general population. With roughly one in fifty people in many populations carrying an SMN1 deletion, and couples who are both carriers facing a one-in-four chance of an affected child in each pregnancy, carrier screening has become a public health priority, and the accuracy of that screening depends entirely on the resolution of the underlying technology.

The study also catalogued the distribution of SMN2, the modifier gene that shapes disease severity. Two copies of SMN2 were the most common configuration at 57.59 percent, one copy accounted for 33.07 percent, and small fractions of individuals carried three or four copies. Notably, 6.10 percent of the screened population carried homozygous SMN2 deletions, a genotype that is not itself disease-causing but eliminates the compensatory buffer that SMN2 provides. Because SMN2 copy number is inversely related to clinical severity in SMA patients, knowing a patient’s SMN2 count helps predict disease course and informs treatment decisions, particularly now that disease-modifying therapies exist for a condition that was, until recently, essentially untreatable.

Four qPCR-confirmed SMA patients served as independent validation cases, and their stories illustrate both the technology’s power and the disease’s variability. Patient 1, a 16-year-old female with severe scoliosis who first showed hand tremors at twelve months, had zero copies of SMN1 and three copies of SMN2 confirmed by all three methods, consistent with SMA type II. Patient 2, a 12-year-old male unable to walk independently at seventeen months, showed the same molecular profile with profound proximal weakness and absent reflexes, also diagnosed as type II. Patient 3, a 17-year-old male with flexion contractures, generalized hypotonia, and complete lower limb paralysis, carried zero SMN1 copies and three SMN2 copies but was classified as type I. Patient 4, a three-month-old male with hypotonia since birth, had zero SMN1 copies and two SMN2 copies, with electromyography revealing nerve fiber damage across multiple limbs, confirming type I disease. No pathogenic point mutations were detected in any patient.

The discordance among patients sharing three SMN2 copies is scientifically telling. Three of the four patients carried the same SMN2 count yet spanned two clinical types, echoing a large analysis of 1,627 SMA patients with three SMN2 copies in which fifteen percent had type I disease, fifty-four percent type II, and thirty-one percent type III. The relationship between copy number and severity is clearly not a simple inverse correlation; even among patients with four or five SMN2 copies, one and five percent respectively still develop type I disease. Whether different SMN2 variants possess distinct functional properties, or whether modifying mutations within SMN2 influence its compensatory capacity, remains an open question that the authors flag for future investigation.

The study’s limitations are candidly acknowledged. Point mutations in SMN1 account for roughly two percent of SMA carrier alleles, yet none were detected in this cohort, a result that may reflect their genuine rarity or a sample size insufficient to capture such infrequent events. Larger-scale studies will be needed to validate the technology’s performance on rare genotypes. Nevertheless, the trajectory is clear: SMA genetic testing has evolved from single-site detection toward whole-gene, haplotype-level analysis, and long-read sequencing has already matured into clinical practice for other monogenic disorders such as thalassemia and congenital adrenal hyperplasia. With its ability to resolve copy numbers, haplotypes, and point mutations in a single assay across one of the human genome’s most treacherous paralogous regions, single-molecule real-time sequencing appears poised to become a cornerstone of SMA diagnosis, carrier screening, and genetic counseling, potentially catching silent carriers that today’s methods routinely miss.

Subject of Research: Clinical application of single-molecule real-time long-read sequencing for spinal muscular atrophy diagnosis and carrier screening

Article Title: Clinical Practice of Single‐Molecule Real‐Time Sequencing in the Diagnosis of Spinal Muscular Atrophy

Article References: Wei, H., Zheng, Y.-W., Zhang, N.-K., Gao, T.-L., Zheng, J.-X., Li, T.-Y., & Hong, S.-R. (2026). Clinical Practice of Single‐Molecule Real‐Time Sequencing in the Diagnosis of Spinal Muscular Atrophy. Molecular Genetics & Genomic Medicine, 14(10), Article e70314. https://doi.org/10.1002/mgg3.70314

Image Credits: AI Generated

DOI: 10.1002/mgg3.70314

Keywords: spinal muscular atrophy, SMRT sequencing, SMN1, SMN2, long-read sequencing, carrier screening, genetic diagnosis, copy number variation, qPCR, MLPA, motor neuron disease, genetic counseling

The research, led by Çağrı Doğan of the Department of Medical Genetics at Ordu University Training and Research Hospital together with pediatric neurologist Seren Aydın of Ondokuz Mayıs University Hospital, examined every consecutive individual tested for DMD variants at their centre between January 2022 and February 2026. Forty-seven individuals passed through the diagnostic pipeline, 29 female and 18 male, with a median age of 12.8 years. The team applied two complementary molecular techniques: multiplex ligation-dependent probe amplification, or MLPA, a copy-number detection method available for 45 of the 47 participants, and whole-gene sequencing, performed in 16 individuals and run in parallel with MLPA in 13. All variants were described against the reference transcript NM_004006.3 and classified according to the 2015 ACMG/AMP standards for sequence variants and the 2020 ACMG/ClinGen standards for copy-number variants.

The headline result is deceptively simple: pathogenic variants were identified in 19 of the 47 individuals. Ten of these were probands, the first affected or index individuals in their families to receive a molecular diagnosis, and nine were heterozygous female carriers discovered through cascade screening, the systematic testing of relatives once a familial variant is known. In index testing, the diagnostic yield was 30.3 percent, or ten of 33 individuals tested, with a 95 percent confidence interval of 17.4 to 47.3 percent. Once a familial variant was established, cascade screening detected carriers in nine of 14 relatives tested, a carrier-detection rate of 64.3 percent with a confidence interval of 38.8 to 83.7 percent. Those figures underline a point that geneticists have long emphasised: identifying one patient can protect an entire extended family.

The variant spectrum itself conformed closely to what has been reported across Türkiye and worldwide. Among the nine families with characterised variants, seven carried deletions and two carried single-nucleotide sequence variants: a nonsense change, NM_004006.3:c.701C>G producing p.(Ser234Ter), and a canonical splice-site variant, c.2803+1G>T, which disrupts the invariant guanine at the first position of an intron and is predicted to derail RNA splicing. Strikingly, six of the nine families carried deletions within the so-called distal hotspot spanning exons 45 to 55, a known deletion-prone region of this extraordinarily large gene. Three of the deletions were in-frame, meaning the reading frame of the dystrophin protein was preserved, and four were out-of-frame, consistent with the classical reading-frame rule that links out-of-frame disruptions to the severe Duchenne phenotype and in-frame disruptions to the milder Becker form. Each deletion was cross-verified against public resources, the Leiden Open Variation Database and the DOVE deletion-out-of-frame evaluation tool.

The study also captured a subtler and less frequently discussed facet of dystrophinopathy: female carriers with biochemical evidence of muscle damage. Four heterozygous women, three of whom were probands in their own right rather than relatives of an affected boy, presented with unexplained hyperCKemia, with creatine kinase values ranging from 776 to 2,816 U/L, but no documented muscle weakness. The one carrier who underwent complete evaluation had normal strength and a normal echocardiogram. This pattern reflects the stochastic nature of X-chromosome inactivation, the process by which female cells randomly silence one X chromosome. When inactivation happens to favour the chromosome carrying the mutant DMD allele, enough dystrophin-deficient muscle fibres can exist to leak creatine kinase into the blood, occasionally producing overt disease in women, including cardiomyopathy, decades before anyone suspects a condition classically associated with boys.

Two of the deletions were apparently de novo, arising fresh in the affected child rather than being inherited from a carrier mother, a well-recognised phenomenon in DMD given the enormous size of the gene and its high spontaneous mutation rate. Equally consequential for treatment planning, none of the probands in this series carried a variant amenable to an approved exon-skipping oligonucleotide. These antisense drugs, including eteplirsen, golodirsen, viltolarsen and casimersen, work by masking specific exons during RNA splicing so that the reading frame is restored and a shorter but partly functional dystrophin protein can be made. Eligibility depends entirely on the exact exon boundaries of the deletion or point mutation, which is why a precise molecular diagnosis is not merely academic but the gateway to genotype-matched therapy.

Technically, the study illustrates the standard two-step architecture of DMD molecular diagnostics. MLPA, first described by Schouten and colleagues in 2002, uses pairs of probes that ligate only when bound to their target sequences and are then amplified, allowing relative quantification of all 79 exons of the DMD gene in a single reaction; it remains the most efficient way to detect the exon deletions and duplications that account for the majority of pathogenic DMD variants. When MLPA is negative, sequencing of the gene captures the point mutations, small insertions and deletions, and splice-site changes that copy-number assays cannot see. Running both methods in parallel, as this centre did in 13 cases, accelerates turnaround. The authors’ adherence to Human Genome Variation Society nomenclature and to consensus ACMG classification frameworks ensures that their variants can be compared directly with entries in international databases such as the TREAT-NMD global mutation registry, which has catalogued more than 7,000 DMD mutations.

The regional significance of the work lies in establishing a baseline. Previous Turkish series, including a 2019 analysis of 260 dystrophinopathy patients and later single-centre surveys, had sketched the national mutation spectrum, but the Central-Eastern Black Sea provinces had remained a blank spot on that map. The new data suggest that the local spectrum is broadly congruent with national and global patterns, dominated by deletions concentrated in the distal hotspot. That congruence matters for genetic counselling: it means clinicians in the region can reasonably apply the established genotype-phenotype correlations and exon-skipping eligibility frameworks without needing region-specific correction, while still sequencing each family’s variant to confirm its exact boundaries.

The authors are careful about the limits of what such a small series can prove. With only ten probands, the reported proportions are descriptive rather than prevalence estimates, and genotype-based phenotype assignment in individual patients requires prospective confirmation with longitudinal follow-up. The retrospective, single-centre design also means the cohort reflects referral patterns and test availability at one hospital rather than systematic population screening. Still, the cascade-screening outcome, nine additional heterozygotes identified from fourteen relatives, is a concrete demonstration of why molecular diagnosis should trigger immediate family testing. Every carrier identified can be offered cardiac surveillance, reproductive counselling including prenatal and preimplantation genetic options, and the knowledge that ultimately saves the next generation of boys in the family from a delayed diagnosis.

For a disease in which early corticosteroid treatment, standardised cardiac and respiratory care, and emerging gene-targeted therapies all depend on catching patients early, the message from Türkiye’s Black Sea coast is clear: find the variant in one child, and the laboratory work has only just begun. The first regional map of DMD variants is now drawn, and its most important lines may be the family trees radiating outward from each proband.

Subject of Research: Mutational spectrum of DMD gene variants and cascade screening for Duchenne and Becker muscular dystrophy in Türkiye

Article Title: Mutational Spectrum of DMD Gene Variants and Cascade Screening Outcomes in the Central-Eastern Black Sea Region of Türkiye: A Single-Centre Retrospective Study

Article References: Doğan, Ç., & Aydın, S. (2026). Mutational Spectrum of DMD Gene Variants and Cascade Screening Outcomes in the Central-Eastern Black Sea Region of Türkiye: A Single-Centre Retrospective Study. Molecular Biology Reports, 53(1), Article 1600. https://doi.org/10.1007/s11033-026-12785-9

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12785-9

Keywords: Duchenne muscular dystrophy, DMD gene, Becker muscular dystrophy, cascade screening, MLPA, female carriers, exon deletions, genetic testing, dystrophinopathy, hyperCKemia, Türkiye, X-chromosome inactivation