Timed rolling and rising tests in Duchenne muscular dystrophy ambulant boys: a feasibility study.

BACKGROUND: Functional activities are extensively used in motor assessments of patients with Duchenne muscular dystrophy. The role of timed items has been reported as an early prognostic factor for disease progression. However, there are two functional activities that are not widely assessed in clinical practice among Duchenne muscular dystrophy patients: rolling and bed rising. This study aimed to investigate whether the 360-degree roll (roll) and supine to sit-to-edge (bed rise) measurements are feasible tools reflecting the functional status of ambulatory DMD children by establishing possible correlations between validated measures: the Vignos Scale (VS), timed rise from floor and the 6-Minute Walk Test (6MWT). METHODS: A total of 32 ambulant boys with DMD were assessed using timed items, the 6MWT and VS. RESULTS: The roll and bed rise are correlated with each other. The 6MWT, the floor rise and VS are correlated with the roll and with the bed rise. CONCLUSIONS: Findings offer preliminary empirical evidence addressing feasibility and safety of roll and bed rise measurements. There is a potential clinical utility of these tests in assessing functional status of DMD ambulant patients.

[Screening for Duchenne muscular dystrophy in newborns in the Ningxia region]. / å®å¤åœ°åŒºæ–°ç”Ÿå„¿æœæ°è‚Œè¥å »ä¸è‰¯ç—‡ç­›æŸ¥.

OBJECTIVES: To evaluate the incidence rate of Duchenne muscular dystrophy (DMD) in the male newborns in the Ningxia region and establish a critical threshold for screening DMD in newborns to distinguish between the normal population and affected individuals. METHODS: A total of 10 000 male newborns were screened using immunofluorescence analysis of creatine kinase isoenzyme concentrations in heel spot dried blood specimens. Newborns with the concentrations higher than the critical threshold were recalled for serum creatine kinase measurements. Genetic testing was performed to confirm diagnosis in cases showing abnormalities. RESULTS: Among the screened 10 000 male newborns, two were confirmed to have DMD through genetic testing, resulting in a preliminary estimated incidence rate of 1/5 000 for male newborns in the Ningxia region. The critical threshold for creatine kinase isoenzyme concentration in newborns in this region was determined to be 468.57 ng/mL. CONCLUSIONS: Screening for DMD in newborns is feasible in the Ningxia region. Early screening, diagnosis, and treatment of DMD can improve the quality of life for affected individuals and help families make informed decisions regarding further pregnancies.

[Efficiency of CNV-seq in detecting fetal DMD gene deletion or duplication in prenatal diagnosis].

Objective: To evaluate the diagnostic efficiency of copy number variation sequencing (CNV-seq) to detect the deletion or duplication of DMD gene in prenatal diagnosis. Methods: A retrospective analysis was carried out on the CNV-seq results of 34 544 fetuses diagnosed in the First People's Hospital of Yunnan Province from January 2018 to July 2023. A total of 156 cases of fetuses were collected, including Group 1:125 cases with family history of Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD), and Group 2:31 cases with no family history but a DMD gene deletion or duplication was detected unexpectedly by CNV-seq. Multiplex ligation-dependent probe amplification (MLPA) was used as a standard method to detect the deletion or duplication. Consistency test was carried out basing on the results of CNV-seq and MLPA of all 156 cases. Results: Comparing to MLPA, CNV-seq had a coincidence rate of 92.3% (144/156) for DMD gene deletion or duplication, with a sensitivity and positive predictive value of 88.2%, with a specificity and negative predictive value of 94.3%, a missed detection rate of 3.8%, and a Kappa value of 0.839. CNV-seq missed 4 cases with deletions and 2 with duplications due to involved fragments less than 100 Kb, among 20 cases of deletions and 6 cases of duplications detected by MLPA in Group 1. In Group 2, the deletions and duplications detected by CNV-seq were 42% (13/31) and 58% (18/31), respectively, in which the percentage of duplication was higher than that in Group 1. Among those 18 cases with duplications, 3 cases with duplication locating in exon 42~67 were likely pathogenic; while 9 cases with duplication covering the 5' or 3' end of the DMD gene, containing exon 1 or 79 and with only one breakpoint within the gene, along with the last 6 cases with duplications locating at chrX: 32650635_32910000 detected only by CNV-seq, which might be judged as variants of uncertain significance. Conclusions: CNV-seq has a good efficiency to detect fetal DMD gene deletion or duplication in prenatal diagnosis, while a further verification test by MLPA is recommended. The duplications on chrX: 32650635_32910000, 5' or 3' end of DMD gene detected by CNV-seq should be carefully verified and assessed because those variants appear to be nonpathogenic polymorphisms.

Comprehensive analysis of genomic complexity in the 5' end coding region of the DMD gene in patients of exons 1-2 duplications based on long-read sequencing.

BACKGROUND: Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are a common type of pathogenic variants in the DMD gene, however, 5' end exonic duplications containing exon 1 are less common. When assessing the pathogenicity of exonic duplications in the DMD gene, consideration must be given to their impact on the reading frame. Traditional molecular methods, such as multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS), are commonly used in clinics. However, they cannot discriminate the precise physical locations of breakpoints and structural features of genomic rearrangement. Long-read sequencing (LRS) can effectively overcome this limitation. RESULTS: We used LRS technology to perform whole genome sequencing on three families and analyze the structural variations of the DMD gene, which involves the duplications of exon 1 and/or exon 2. Two distinct variant types encompassing exon 1 in the DMD Dp427m isoform and/or Dp427c isoform are identified, which have been infrequently reported previously. In pedigree 1, the male individuals harboring duplication variant of consecutive exons 1-2 in the DMD canonical transcript (Dp427m) and exon 1 in the Dp427c transcript are normal, indicating the variant is likely benign. In pedigree 3, the patient carries complex SVs involving exon 1 of the DMD Dp427c transcript showing an obvious phenotype. The locations of the breakpoints and the characteristics of structural variants (SVs) are identified by LRS, enabling the classification of the variants' pathogenicity. CONCLUSIONS: Our research sheds light on the complexity of DMD variants encompassing Dp427c/Dp427m promoter regions and emphasizes the importance of cautious interpretation when assessing the pathogenicity of DMD 5' end exonic duplications, particularly in carrier screening scenarios without an affected proband.

Draft Guidance for Industry Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, and Related Dystrophinopathies - Developing Potential Treatments for the Entire Spectrum of Disease.

Background: Duchenne muscular dystrophy (DMD) and related dystrophinopathies are neuromuscular conditions with great unmet medical needs that require the development of effective medical treatments. Objective: To aid sponsors in clinical development of drugs and therapeutic biological products for treating DMD across the disease spectrum by integrating advancements, patient registries, natural history studies, and more into a comprehensive guidance. Methods: This guidance emerged from collaboration between the FDA, the Duchenne community, and industry stakeholders. It entailed a structured approach, involving multiple committees and boards. From its inception in 2014, the guidance underwent revisions incorporating insights from gene therapy studies, cardiac function research, and innovative clinical trial designs. Results: The guidance provides a deeper understanding of DMD and its variants, focusing on patient engagement, diagnostic criteria, natural history, biomarkers, and clinical trials. It underscores patient-focused drug development, the significance of dystrophin as a biomarker, and the pivotal role of magnetic resonance imaging in assessing disease progression. Additionally, the guidance addresses cardiomyopathy's prominence in DMD and the burgeoning field of gene therapy. Conclusions: The updated guidance offers a comprehensive understanding of DMD, emphasizing patient-centric approaches, innovative trial designs, and the importance of biomarkers. The focus on cardiomyopathy and gene therapy signifies the evolving realm of DMD research. It acts as a crucial roadmap for sponsors, potentially leading to improved treatments for DMD.

[Characteristics and changes of cardiac injury with age in children of Duchenne muscular dystrophy: a prospective cohort study].

Objective: To explore the characteristics and changes of cardiac injury with age in Duchenne muscular dystrophy (DMD) and its clinical significance. Methods: A prospective cohort study was conducted. The 215 patients diagnosed with DMD in West China Second Hospital from January 2019 to November 2022 and aged from 6 to 18 years were enrolled. Their clinical data, myocardial injury markers, routine electrocardiogram, cardiac magnetic resonance (CMR) and echocardiography were collected. The patients were divided into five age groups: 6-<8, 8-<10, 10-<12, 12-<14 and 14-18 years of age, and matched with healthy boys respectively. Independent sample t test or Mann-Whitney U test was used to compare the clinical data and CMR indexes between DMD patients and controls in all age subgroups, and to compare the value of left ventricular ejection fraction (LVEF) measured by echocardiography and CMR in each subgroup of DMD patitents. Pearson correlation analysis or Spearman correlation analysis was used to explore the relation between the CMR indexes and age in DMD patients. Results: A total of 215 patients with DMD (all male) and 122 healthy boys were included in the study. There were 75 DMD patients and 23 controls in 6-<8 years of age group, 77 DMD and 28 controls in 8-<10 years of age group, 39 DMD and 23 controls in 10-<12 years of age group, 10 DMD and 31 controls in the 12-<14 years of age group, and 14 DMD and 17 controls in 14-18 years of age group. In the DMD patients, the older the age, the lower the levels of creatine kinase (CK) and creatine kinase isoenzyme (CK-MB). In the 6-<8 years of age group, the CK level was 10 760 (7 800, 15 757) U/L, while in the group of 14-18 years of age, it was 2 369 (1 480, 6 944) U/L. As for CK-MB, it was (189±17) µg/L in the 6-<8 years of age group and (62±16) µg/L in the 14-18 years of age group. Cardiac troponin I remained unchanged in <12 years of age groups, but significantly increased in 12-<14 years of age group, reaching the highest value of 0.112 (0.006, 0.085) µg/L. In the DMD patients, the older the age, the higher the proportion of abnormal electrocardiogram (ECG). In the 6-<8 years of age group, the proportion is 29.3% (22/75), while in the 14-18 years of age group, it was 10/14. Correlation analysis showed that the left ventricular end-diastolic volume index was positively related with age (r=0.18, P=0.015), and the left ventricular stroke volume index and cardiac output index were negatively related with age (r=-0.34 and -0.31, respectively, both P<0.001). In the DMD patients, the older the age, the lower LVEF, with the LVEF decreasing to (49.3±3.1)% in the 14-18 years of age group. The LVEF of DMD cases was significantly lower than that of controls in the age subgroups of 8-<10, 10-<12, 12-<14 and 14-18 years of age groups ((57.9±5.2) % vs. (63.6±0.8)%, 60.7% (55.9%, 61.9%) vs. 63.7% (60.2%, 66.0%), 57.1% (51.8%, 63.4%) vs. 62.1 % (59.5%, 64.5)%, (49.3±3.1) % vs. (61.6±1.3)%, respectively; all P<0.01). In the DMD patients, the older the age, the higher the proportion of positive late gadolinium enhancement (LGE). In the 6-<8 years of age group, it was 22% (11/51), in the 12-<14 years of age group, it was 13/14, and in the 14-18 years of age group, all DMD showed positive LGE. The value of LVEF of DMD cases measured by echocardiography was significantly higher than that measured by CMR in 6-<8 years of age group and 8-<10 years of age group (63.2% (60.1%, 66.4%) vs. 59.1 % (55.4%, 62.9%), and (62.8±5.2) % vs. (57.9±5.2)%, all P<0.001). Conclusion: DMD patients develop cardiac injury in the early stage of the disease, and the incidence of cardiac damage gradually increases with both age and the progression of disease.

Patient's perspective about research landscape for rare diseases in India.

I am writing this piece as the parent of a son diagnosed with Duchenne muscular dystrophy, a severely debilitating disease that not only impairs skeletal muscles of the limbs but is also life-threatening due to progressive weakening of the cardiac and diaphragm muscles. I have traversed the harrowing diagnostic, treatment, and management odyssey of a typical rare disease (RD) patient in India.

Diagnostic Accuracy of Creatine Kinase Isoenzyme-MM Test in Newborn Screening for Duchenne Muscular Dystrophy: A Systematic Review and Meta-Analysis.

BACKGROUND: To systematically evaluate the diagnostic accuracy of the creatine kinase isoenzyme-MM (CK-MM) test in newborn screening for Duchenne muscular dystrophy (DMD). METHODS: A comprehensive literature search was conducted up to October 31, 2022, in PubMed, Embase, Cochrane Library, Web of Science, and Scopus Database. To evaluate the diagnostic value, the sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), area under the curve (AUC), and Q∗ index were pooled. Threshold effect followed by subgroup analysis and meta-regression were performed to explore the source of heterogeneity. Sensitivity analysis was used to verify the robustness of the findings. RESULTS: A total seven studies with 248,853 newborns was included in our meta-analysis. The pooled SEN and SPE were 1.00 (95% confidence interval [CI]: 0.89∼1.00) and 1.00 (95% CI: 1.00 to 1.00), respectively; the PLR and NLR were 1004.59 (95% CI: 251.37∼4014.91) and 0.13 (95% CI: 0.05∼0.34), respectively; the DOR was 877.96 (95% CI: 983.24∼78,366.32); the AUC and Q index were 0.8683 and 0.9326, respectively. Sensitivity analysis showed that two studies had an impact on the pooled results and mainly contributed to the heterogeneity. CONCLUSIONS: CK-MM test demonstrated high accuracy in newborn screening for DMD and may be a valuable alternative in the early diagnosis of the disease followed by confirmatory genetic testing.

[Chinese guidelines on the diagnosis of dystrophinopathy].

Dystrophinopathy refers to a group of X-linked recessive myopathies that primarily affect skeletal and/or cardiac muscle caused by pathogenic variants in the dystrophin-encoding DMD gene, including Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy. The broad and complex spectrum of pathogenic DMD variants complicates the diagnosis and clinical classification in some patients. The precise genetic diagnosis is of great significance for the clinical diagnosis and treatment, multidisciplinary management, genetic counseling, prenatal diagnosis, and selection of gene therapy in dystrophinopathy. The present guideline is primarily based on the research advances in dystrophinopathy. Meanwhile, the foreign and domestic clinical guidelines or consensus for dystrophinopathy were referenced to put forward 18 recommendations and reach a consensus on the clinical manifestations, genetic basis, clinical diagnosis and classification, genetic diagnosis, and clinical genetic counseling of dystrophinopathy. This guideline aims to standardize and optimize the diagnostic process and reduce the diagnostic difficulty of patients with dystrophinopathy. In addition, this guideline provides some practical reference for clinicians and government staff.

The Early Care (0-3 Years) In Duchenne Muscular Dystrophy Meeting Report.

Objective: This report summarizes the key discussions from the "Early Care (0-3 years) in Duchenne Muscular Dystrophy" meeting, which aimed to address the challenges and opportunities in the diagnosis and care of Duchenne muscular dystrophy (DMD) and female carriers within the 0-3-year age group. Methods: The meeting brought together experts and healthcare providers who shared insights, discussed advancements in DMD care, and identified research needs. Presentations covered diagnostic challenges, approved therapies, clinical trials, identification of young female carriers, and the importance of clinical care and support for families. Results: The meeting highlighted the importance of timely diagnosis and the lack of evidence-based guidelines for the care of children with DMD aged 0-3 years. Diagnostic challenges were discussed, including delays in receiving a DMD diagnosis and disparities based on ethnicity. The potential benefits and process of newborn screening were addressed.Approved therapeutic interventions, such as corticosteroids and exon-skipping drugs, were explored, with studies indicating the potential benefits of early initiation of corticosteroid therapy and the safety of exon-skipping drugs in DMD. Clinical trials involving infants and young boys were discussed, focusing on drugs like ataluren, vamorolone, and gene therapies.The meeting emphasized the importance of clinical care and support for families, including comprehensive information provision, early intervention services, and individualized support. The identification and care of young female carriers were also addressed. Conclusion: The meeting provided a platform for experts and healthcare providers to discuss and identify key aspects of early care for children with DMD aged 0-3 years. The meeting emphasized the need for early diagnosis, evidence-based guidelines, and comprehensive care and support for affected children and their families. Further research, collaboration, and the development of consensus guidelines are needed to improve early diagnosis, treatment, and outcomes in this population.

Burden of Disease of Duchenne Muscular Dystrophy in Denmark - A National Register-Based Study of Individuals with Duchenne Muscular Dystrophy and their Closest Relatives.

Background: Duchenne Muscular Dystrophy (DMD) is a progressive genetic disease with a prevalence of 1 per 3,600-6,000 male births. Individuals with DMD are typically diagnosed at age 4-7 years; median survival is 30 years. They require multidisciplinary care, personal assistance, and often special education. Objective: The aim was to assess the burden of disease in DMD in Denmark. This includes incidence, prevalence, use of healthcare services, labour market participation, educational outcomes, and overall attributable costs due to DMD. Impact on the closest relatives (siblings and parents) was also investigated. Methods: The comprehensive Danish national health and administrative registers were used to assess the burden of disease following individuals with DMD and closest relatives from five years before, and up to 20 years after DMD diagnosis. Individuals with DMD (and relatives) from 1994-2021 were included. All outcomes were compared to matched control groups without the disease drawn from the Danish population. Results: 213 unique individuals with DMD were identified. They had lower grades in school, required more special education and more healthcare and home care compared to their control group. The extra costs of special education summed to EUR 180,900 over the course of 11 years elementary school. They had an annual average productivity loss of EUR 20,200 between the age of 18 to 30. The extra healthcare costs of DMD in the 20 years after diagnosis were estimated to EUR 1,524,000. If an individual with DMD lives to be 30, total extra costs sum to EUR 2,365,800. Conclusions: Using national register data this study presented detailed results on the burden of disease of DMD, including impact on closest relatives. With 60 additional hospital admissions and 200 extra outpatient contacts in 20 years healthcare costs, but also costs of home care and special education, increases as disease progresses.

Advancing Biomarker Discovery and Therapeutic Targets in Duchenne Muscular Dystrophy: A Comprehensive Review.

Mounting evidence underscores the intricate interplay between the immune system and skeletal muscles in Duchenne muscular dystrophy (DMD), as well as during regular muscle regeneration. While immune cell infiltration into skeletal muscles stands out as a prominent feature in the disease pathophysiology, a myriad of secondary defects involving metabolic and inflammatory pathways persist, with the key players yet to be fully elucidated. Steroids, currently the sole effective therapy for delaying onset and symptom control, come with adverse side effects, limiting their widespread use. Preliminary evidence spotlighting the distinctive features of T cell profiling in DMD prompts the immuno-characterization of circulating cells. A molecular analysis of their transcriptome and secretome holds the promise of identifying a subpopulation of cells suitable as disease biomarkers. Furthermore, it provides a gateway to unraveling new pathological pathways and pinpointing potential therapeutic targets. Simultaneously, the last decade has witnessed the emergence of novel approaches. The development and equilibrium of both innate and adaptive immune systems are intricately linked to the gut microbiota. Modulating microbiota-derived metabolites could potentially exacerbate muscle damage through immune system activation. Concurrently, genome sequencing has conferred clinical utility for rare disease diagnosis since innovative methodologies have been deployed to interpret the functional consequences of genomic variations. Despite numerous genes falling short as clinical targets for MD, the exploration of Tdark genes holds promise for unearthing novel and uncharted therapeutic insights. In the quest to expedite the translation of fundamental knowledge into clinical applications, the identification of novel biomarkers and disease targets is paramount. This initiative not only advances our understanding but also paves the way for the design of innovative therapeutic strategies, contributing to enhanced care for individuals grappling with these incapacitating diseases.

Neurodiversity, treatment compliance and survival in adults with Duchenne muscular dystrophy: a single-centre retrospective cohort review.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy worldwide. With increasing survival, there is now a greater awareness of associated neurodevelopmental co-morbidities. Despite this, there is currently a limited understanding of how these co-morbidities might potentially impact on health outcomes. This study reviewed the characteristics of 37 adults with DMD who died between 2011 and 2022. The median age of death was 22.25 years, and those with neurodevelopmental co-morbidities had statistically poorer survival than those without a neurodevelopmental disorder. Notably, the proportion of patients within this cohort with a diagnosis of a neurodevelopmental disorder was higher than previously reported studies in living cohorts. This study suggests that patients with a co-morbid neurodevelopmental disorder may have worse health outcomes than those who do not.

[Analysis of DMD gene variants in a single center].

Objective: To investigate the DMD genetic variants of the Chinese population with Duchenne (DMD) and Becker muscular dystrophies (BMD). Methods: A cross-sectional study was conducted on 2 690 unrelated patients with DMD and BMD aged 0-18 who visited the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from January 2005 to February 2022. The clinical data, such as gender, age, clinical manifestations, and address, were collected. Multiplex ligation-dependent probe amplification, next generation sequencing panel, Sanger sequencing, and PCR amplification were used to detect the variants of the DMD gene in the patients, whose clinical information and gene detection results were descriptively analyzed. Results: The 2 690 patients included 2 648 males and 42 females, with an age of 6.0 (4.0, 9.0) years. The serum creatine kinase increased in all patients. Pathogenic DMD gene variants were detected in the 2 618 patients, including 1 875 cases (71.6%) large deletions, 231 cases (8.8%) duplications, and 512 cases (19.6%) small variants. Among the deletion variants, the deletion of 3 exons was the most common, accounting for 15.4% (288/1 875); and hotspot deletion involved exons 45 to 50, accounting for 6.3% (119/1 875). Exon 2 was the most common type duplication region, accounting for 13.0% (30/231). Small variants were distributed in all 79 exons of the DMD gene, with no hotspots. In addition, the 46 small variants were previously unreported. Conclusion: Exon deletion is the most common type of DMD gene variant, followed by small variants and exon duplication.

Social cognition in DMD and BMD dystrophinopathies: A cross-sectional preliminary study.

Objective: The dystrophinopathies called Duchenne and Becker muscular dystrophies (DMD/BMD) are rare, progressive, incurable, and life-limiting paediatric-onset neuromuscular diseases. These diseases have long been associated with specific neuropsychological deficits. However, the performance of these patients in the social cognition domain has not been properly investigated. Thus, the main objective of this study was to compare the performance on social cognition between DMD/BMD patients and healthy age-matched boys. Method: This cross-sectional study included 20 DMD/BMD children and adolescents and 20 healthy controls. The protocol included the Social Perception Domain of the NEPSY-II, the Reading the Mind in the Eyes Test - Child and Happé's Strange Stories test. General intelligence was controlled to eliminate the possible influence of covariables. All the assessments were performed remotely. Results: Most social cognition tasks were worse in patients with DMD/BMD than in matched healthy controls. These differences remained even after controlling for the general intelligence variable, with the exception of Total Disgust Errors (F = 1.462, p = .234, η2p= .038) and Verbal task (F = 1.820, p = .185, η2p= .047) scores from the NEPSY-II. Conclusions: This is the first study to demonstrate that the neuropsychological domain of social cognition is impaired in DMD/BMD patients, independent of the level of general intelligence. Screening assessments in DMD/BMD patients should be promoted to allow social cognition difficulties to be detected at an early stage to enhance patients' quality of life and social development.

Early assessment of cardiomyopathy in Duchenne patients by means of longitudinal strain echocardiography.

INTRODUCTION: The diagnosis of Duchenne-linked cardiomyopathy may be challenging. Conventional echocardiographic measurements typically show deterioration beyond the second decade. Global longitudinal strain has been proposed as an earlier marker than left ventricular ejection fraction. MATERIAL AND METHODS: A prospective, observational, cross-sectional, case-control study was carried out. Both Duchenne patients and control subjects underwent transthoracic echocardiogram in order to assess left ventricle function. Bayesian linear regression was the main tool for inference. Age effects were parameterised by means of a spline function because of its simplicity to characterise continuous variables and smooth contributions. The posterior distribution of the marginal age effects was used to assess the earliest age of deterioration of each marker. RESULTS: Sixteen Duchenne patients and twenty-two healthy male subjects were enrolled. On overage, cardiac function measures were found for ejection fraction and longitudinal strain among different groups. Age effects on global longitudinal strain are more reliably found at ages of 6 and above, while ejection fraction starts to deteriorate at an older age. Progressive left ventricular dysfunction in Duchenne patients is one of the key issues and starts at an early age with subtle symptoms. CONCLUSION: This cross-sectional study provides supporting evidence that global longitudinal strain is an earlier marker of disease progression than ejection fraction in Duchenne patients.

Two years of newborn screening for Duchenne muscular dystrophy as a part of the statewide Early Check research program in North Carolina.

PURPOSE: Current and emerging treatments for Duchenne muscular dystrophy (DMD) position DMD as a candidate condition for newborn screening (NBS). In anticipation of the nomination of DMD for universal NBS, we conducted a prospective study under the Early Check voluntary NBS research program in North Carolina, United States. METHODS: We performed screening for creatine kinase-MM (CK-MM), a biomarker of muscle damage, on residual routine newborn dried blood spots (DBS) from participating newborns. Total creatine kinase testing and next generation sequencing of an 86-neuromuscular gene panel that included DMD were offered to parents of newborns who screened positive. Bivariate and multivariable analyses were performed to assess effects of biological and demographic predictors on CK-MM levels in DBS. RESULTS: We screened 13,354 newborns and identified 2 males with DMD. The provisional 1626 ng/mL cutoff was raised to 2032 ng/mL to improve specificity, and additional cutoffs (900 and 360 ng/mL) were implemented to improve sensitivity for older and low-birthweight newborns. CONCLUSION: Population-scale screening for elevated CK-MM in DBS is a feasible approach to identify newborns with DMD. Inclusion of birthweight- and age-specific cutoffs, repeat creatine kinase testing after 72 hours of age, and DMD sequencing improve sensitivity and specificity of screening.

Application of a Multiplex Ligation-Dependent Probe Amplification-Based Next-Generation Sequencing Approach for the Detection of Pathogenesis of Duchenne Muscular Dystrophy and Spinal Muscular Atrophy Caused by Copy Number Aberrations.

Both Duchenne muscular dystrophy (DMD; OMIM no. 310200) and spinal muscular atrophy (SMA; OMIM no. 253300/253550/253400/271150) are genetic disorders characterized by progressive muscle degeneration and weakness. Genetic copy number aberrations in the pathogenetic genes DMD and SMN1 lead to alterations in functional proteins, resulting in DMD and SMA, respectively. Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for the detection of common copy number aberrations (CNAs), including DMD and SMN1 deletions, both of which are associated with poor clinical outcomes. However, traditional MLPA assays only accommodate a maximum of 60 MLPA probes per test. To increase the number of targeted sequences in one assay, an MLPA-based next-generation sequencing (NGS) assay has been developed that is based on the standard MLPA procedure, allows high-throughput screening for a large number of fragments and samples by integrating additional indices for detection, and can be analyzed on all Illumina NGS platforms.

Exploring factors impacting haplotype-based noninvasive prenatal diagnosis for single-gene recessive disorders.

Haplotype-based noninvasive prenatal diagnosis (NIPD) is applicable for various recessive single-gene disorders in proband families. However, a comprehensive exploration of critical factors influencing the assay performance, such as fetal fraction, informative single nucleotide polymorphism (SNP) count, and recombination events, has yet to be performed. It is critical to identify key factors affecting NIPD performance, including its accuracy and success rate, and their impact on clinical diagnostics to guide clinical practice. We conducted a prospective study, recruiting 219 proband families with singleton pregnancies at risk for eight recessive single-gene disorders (Duchenne muscular dystrophy, spinal muscular atrophy, phenylketonuria, methylmalonic acidemia, hemophilia A, hemophilia B, non-syndromic hearing loss, and congenital adrenal hyperplasia) at 7-14 weeks of gestation. Haplotype-based NIPD was performed by evaluating the relative haplotype dosage (RHDO) in maternal circulation, and the results were validated via invasive prenatal diagnosis or newborn follow-ups. Among the 219 families, the median gestational age at first blood draw was 8+5 weeks. Initial testing succeeded for 190 families and failed for 29 due to low fetal fraction (16), insufficient informative SNPs (9), and homologous recombination near pathogenic variation (4). Among low fetal fraction families, successful testing was achieved for 11 cases after a redraw, while 5 remained inconclusive. Test failures linked to insufficient informative SNPs correlated with linkage disequilibrium near the genes, with F8 and MMUT exhibiting the highest associated failure rates (14.3% and 25%, respectively). Homologous recombination was relatively frequent around the DMD and SMN1 genes (8.8% and 4.8%, respectively) but led to detection failure in only 44.4% (4/9) of such cases. All NIPD results from the 201 successful families were consistent with invasive diagnostic findings or newborn follow-up. Fetal fraction, informative SNPs count, and homologous recombination are pivotal to NIPD performance. Redrawing blood effectively improves the success rate for low fetal fraction samples. However, informative SNPs count and homologous recombination rates vary significantly across genes, necessitating careful consideration in clinical practice. We have designed an in silico method based on linkage disequilibrium data to predict the number of informative SNPs. This can identify genomic regions where there might be an insufficient number of SNPs, thereby guiding panel design. With these factors properly accounted for, NIPD is highly accurate and reliable.

Prenatal diagnosis of 1408 foetuses at risk of DMD/BMD by MLPA and Sanger sequencing combined with STR linkage analysis.

OBJECTIVE: This study is a retrospective analysis of the prenatal genetic diagnosis results of 1408 foetuses at high risk of DMD/BMD to provide information for clinical genetic counselling. BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder characterized by skeletal and cardiac muscle weakness. With the deepening of disease research, some treatments have been applied in clinics. Therefore, early and accurate prenatal diagnosis can inform pregnancy choices for high-risk families. METHODS: A total of 1316 unrelated DMD/BMD families with confirmed genetic diagnoses were recruited from the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University. Prenatal diagnosis of 1408 high-risk foetuses was performed by MLPA and Sanger sequencing combined with STR linkage analysis for all families. RESULTS: Among the 1316 families, large deletions, duplications, and small variants of the DMD gene accounted for 70.4% (927/1316), 8.2% (108/1316), and 21.4% (281/1316), respectively. Among 1316 mothers, 863 (65.6%) were carriers, and 453 (34.4%) were not carriers. The rate of de novo variants was 34.4% (453/1316) in our study. In addition, gonadal mosaicism was observed in 11 pregnant females. Prenatal diagnosis was provided for 1408 high-risk foetuses; 282 foetuses were identified as male patients, 219 foetuses were female carriers, and the remainder had normal genetics. The results of prenatal diagnosis were consistent with the results of follow-up. CONCLUSIONS: Accurate and rapid prenatal diagnosis can be achieved using MLPA, Sanger sequencing, and STR linkage analysis. Furthermore, germline mosaicism in DMD should not be ignored; considering this, a prenatal diagnosis for all pregnant women with a family history of DMD/BMD regardless of whether they carried disease-causing variants is proposed. Genetic counselling and targeted prenatal diagnosis will continue to be a cornerstone of DMD/BMD family management in the future.