Newborn screening for Duchenne muscular dystrophy: A two‐year pilot study

Duchenne muscular dystrophy (DMD), a progressive X-linked neuromuscular disorder, has an estimated worldwide incidence of 1:3500 male births. Currently, there are no curative treatments and the mean age of diagnosis is 5 years. In addition, subsequent pregnancies frequently occur before a diagnosis is made in an index case. An 'opt in' screening programme was introduced in Wales in 1990 with the aim to: reduce the diagnostic delay, permit reproductive choice and allow planning of the care of the affected boy. Newborn bloodspots were collected routinely as part of the Wales newborn screening programme. Specific consent was obtained for this test separately from the other tests. During the 21-year period, 369,780 bloodspot cards were received from male infants, of these 343,170 (92.8%) were screened using a bloodspot creatine kinase (CK) assay following parental consent. A total of 145 cases had a raised CK activity (≥250 U/l) and at follow-up, at 6-8 weeks of age, 79 cases had a normal serum CK (false-positive rate 0.023%) and 66 cases had an elevated serum CK. DMD was confirmed in 56 cases by genotyping/muscle biopsy studies, Becker muscular dystrophy in 5 cases and other rarer forms of muscular dystrophy in 5 cases. This long-term study has so far identified 13 false-negative cases. The incidence of DMD in Wales of 1:5136 during this period is lower than that of 1:4046 before commencement of screening in Wales. Screening has reduced the diagnostic delay enabling reproductive choice for parents of affected boys and earlier administration of current therapies.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disorder with a birth prevalence of 19.8:100,000 males worldwide. Elevated concentration of the muscle enzyme creatine kinase-MM (CK-MM) allows for presymptomatic screening of newborns using Dried Blood Spots (DBS). We evaluated imprecision and carryover of the FDA-approved PerkinElmer GSP Neonatal CK-MM kit over multiple runs, days, and operators, followed by quantification of CK-MM loss in stored newborn, contrived, and non-newborn patient DBS resulting from exposure to ambient versus low humidity (50-day trial), and high humidity and high temperature (8-day trial). Imprecision %CV was ≤14% for all verification comparisons and over 6 months of testing. On average, the mean CK-MM recovery after 50 days was >80% of initial concentration for all sample types stored in low humidity and <80% in ambient humidity. After 8 days of storage in high humidity and high temperature, the mean recovery for newborn samples was <80%. Verification results for the GSP Neonatal CK-MM assay were concordant with kit parameters and the assay performed consistently over 6 months. CK-MM degradation in ambient storage can be mitigated by reducing exposure to humidity. Assessment of DBS shipping and storage conditions is recommended prior to implementing DMD screening.

BackgroundAt present, Multiplex ligation-dependent probe amplification (MLPA) and exome sequencing are common gene detection methods in patients with Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD), but they can not cover the whole-genome sequence of the DMD gene. In this study, the whole genome capture of the DMD gene and next-generation sequencing (NGS) technology were used to detect the patients with DMD/BMD in Southwest China, to clarify the application value of this technology and further study the gene variant spectrum.MethodsFrom 2017 to 2020, 51 unrelated patients with DMD/BMD in southwestern China were clinically diagnosed at West China Second University Hospital of Sichuan University (Chengdu, China). The whole-genome of the DMD gene was captured from the peripheral blood of all patients, and next-generation sequencing was performed. Large copy number variants (CNVs) in the exon regions of the DMD gene were verified through MLPA, and small variations (such as single nucleotide variation and < 50 bp fragment insertions/deletions) were validated using Sanger sequencing.ResultsAmong the 51 patients, 49 (96.1% [49/51]) had pathogenic or likely pathogenic variants in the DMD gene. Among the 49 positive samples, 17 patients (34.7% [17/49]) had CNVs in the exon regions and 32 patients (65.3% [32/49]) had small variations. A total of six novel variants were identified: c.10916_10917del, c.1790T>A, c.1842del, c.5015del, c.5791_5792insCA, and exons 38–50 duplication.ConclusionsPathogenic or likely pathogenic variants of the DMD gene were detected in 49 patients (96.1% [49/51]), of which 6 variants (12.2% [6/49]) had not been previously reported. This study confirmed the value of NGS-based targeted sequencing for the DMD gene expanding the spectrum of variants in DMD, which may provide effective genetic counseling and prenatal diagnosis for families.

The dystrophinopathies encompass the phenotypically variable forms of muscular dystrophy caused by pathogenic variants in the DMD gene. The dystrophinopathies include the most common inherited muscular dystrophy among 46,XY individuals, Duchenne muscular dystrophy, as well as Becker muscular dystrophy and other less common phenotypic variants. With increased access to and utilization of genetic testing in the diagnostic and carrier setting, genetic counselors and clinicians in diverse specialty areas may care for individuals with and carriers of dystrophinopathy. This practice resource was developed as a tool for genetic counselors and other health care professionals to support counseling regarding dystrophinopathies, including diagnosis, health risks and management, psychosocial needs, reproductive options, clinical trials, and treatment. Genetic testing efforts have enabled genotype/phenotype correlation in the dystrophinopathies, but have also revealed unexpected findings, further complicating genetic counseling for this group of conditions. Additionally, the therapeutic landscape for dystrophinopathies has dramatically changed with several FDA-approved therapeutics, an expansive research pathway, and numerous clinical trials. Genotype-phenotype correlations are especially complex and genetic counselors' unique skill sets are useful in exploring and explaining this to families. Given the recent advances in diagnostic testing and therapeutics related to dystrophinopathies, this practice resource is a timely update for genetic counselors and other healthcare professionals involved in the diagnosis and care of individuals with dystrophinopathies.

Both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations of the X-linked dystrophin gene. BMD patients are less affected clinically than DMD patients. We present five patients with a diagnosis of BMD. First, two identical twins, with a deletion of exon 48 of the dystrophin gene, who experienced prominent muscle cramps from the age of three. The histopathological examination of muscle biopsies of these two twins revealed only very slight muscle fiber alterations. Second, two brothers who displayed marked, unusual intrafamilial variability of the clinical picture as well as showing a new point mutation in the dystrophin gene. And finally, a fifth boy who displayed a new point mutation in the dystrophin gene. Although he was clinically asymptomatic at the age of 15 and muscle biopsy only showed very minor myopathic signs, serum Creatine Kinase (CK) levels had been considerably elevated for years. Taken together, these cases add to the spectrum of marked discrepancies in clinical, histopathological and molecular genetic findings in BMD.

Combination of TREC Measurement and Next-Generation Sequencing in Newborn Screening for Severe Combined Immunodeficiency: A Pilot Program in Japan

Background Patients with Duchenne muscular dystrophy (DMD) develop cardiomyopathy (CM) at an earlier age compared to Becker muscular dystrophy (BMD), but the age of onset within each diagnosis is variable. A method to predict CM onset associated with dystrophinopathy could alter therapeutic approaches and improve outcomes. Shortened post-contrast T1 relaxation times are an early marker of myocardial fibrosis and are abnormal in patients with DMD as described in our previous work. To our knowledge, T1 mapping has not been evaluated in BMD patients. We hypothesized that patients with BMD would also have abnormal T1 times and that these abnormalities would precede LV dysfunction. Methods Twenty-six CMR scans from these 2 dystrophinopathy populations (N=5 BMD and N=21 DMD) were compared with 10 CMR scans from control patients without cardiovascular disease. T1 maps were created from the Look-Locker sequence, obtained 10 minutes after gadolinium injection, using MRMap. Using MatLab, T1 times were obtained for every voxel in 6 standard myocardial segments in the short axis at the level of the papillary muscles. Mean T1 times were compared between BMD, DMD and controls using a Kruskal Wallis test. A subset of patients with normal LVEF and dystrophinopathy (BMD or DMD) was compared to controls using a Mann-Whitney U test. Results The mean age of patients was 25.4 ± 8.1 years in the BMD group, 15.7 ± 4.3 years in the DMD group, and 16.9 ± 1.3 years in controls. One patient with BMD and 13 patients with DMD had LVEF < 55%. Two patients from both BMD and DMD had LV dilatation. BMD and DMD patients had significantly shorter mean postcontrast T1 compared with controls (BMD: 355 ms, 95% CI (314, 395), DMD:357 ms, 95% CI (323, 390), control: 420 ms, 95% CI (389, 451), p=0.035) (Figure 1). The subset of BMD and DMD patients and normal LVEF also had a significant decrease in post-contrast T1 when compared to controls (-52.4 ms, 95% CI (-7.2, -97.5), p=0.018) (Figure 2). Conclusions Myocardial post-contrast T1 relaxation times in BMD and DMD are significantly shorter than in controls. They remain significantly decreased in the subset of patients with normal LVEF, suggesting that CMR may have prognostic utility in detecting subclinical myocardial damage associated with loss of dystrophin prior to a decline in LVEF. Further longitudinal study is necessary to evaluate whether T1 times can predict future onset of CM in BMD and DMD. Funding none.

Event Abstract Back to Event Pilot study for the neonatal screening of congenital T cell lymphopenias Isaura P. Sánchez1, 2*, Jose L. Franco2, Andres A. Arias2, Julio C. Orrego2, Mónica L. Giraldo2, Sebastian Gutierrez Hincapie2, Frank D. Rojo1, Enrique M. Velásquez3, Augusto Quevedo3, Antonio Condino-Neto4 and Claudia M. Trujillo2 1 Corporación Universitaria Remington, Colombia 2 Universidad de Antioquia, Colombia 3 Universidad de Antioquia, Colombia 4 University of São Paulo, Brazil Introduction: T-cell lymphopenias involved at least 24 conditions characterized by a marked decrease or absence of T lymphocytes in peripheral blood (PB). The frequency of these diseases vary from 1:3000 live births for DiGeorge Syndrome (DGS, the most common cause of T-cell lymphopenia) to 1:33.000 for Severe Combined Immunodeficiency (SCID), the most life-threatening T-cell lymphopenia. Although SCID and other severe T cell lymphopenias fulfill the requirements to be included in newborn screening (NBS) programs, these are performed neither worldwide nor in Colombia. T-cell lymphopenias NBS would allow promptly intervention of such conditions through haematopoietic stem cell transplantation (HSCT), preventing severe infections, organ damage, long hospitalization periods, expensive medication and death, which cause tremendous costs for our health care system. T-cell lymphopenias NBS can be easily implemented by quantifying T-cell receptor excision circles (TRECs). The main goal from our study is to lead the implementation of a NBS program for T-cell lymphopenias in Medellín, and at long term, to shape a laboratory network for the prenatal and neonatal diagnosis of these conditions in Colombia based in formally established public health acts about the issue. Methods: This pilot study will conform a network for sample collection with several Medellín Neonatology Units. Quantification of TRECs will be perform by real-time quantitative polymerase chain reaction (qPCR) using DNA isolated from filter paper dried blood spots collected from the heel of 100 newborns. Results: Our proposal was well received by five neonatologist units from Medellin and we are currently applying for the IRB approval. We have also advanced in comparing the TRECs costs using commercial kits with available research-used-only (RUO) reagents already established for the NBS program according to the Jeffrey Modell Centers Network (JMCN, calculated in USD 5 in the USA per test). The use of the kit increases costs by 21-fold. These data emphasize the need of adjusting the protocols for standardizing the TRECs in our country according to the JMCN. We also extrapolate the cost-effective balance of the TRECs testing in Medellin. Considering that the 2013 live birth number in our city was calculated in 27,569 (according to Departamento Administrativo Nacional de Estadística-DANE) and assuming that the DGS incidence in the city is similar to that calculated in the USA, the NBS would detect approximately 9 cases per year. The cost of the NBS program, taking into account an individual screening value of USD 5 in the USA, would be USD 137,845. Taking into account that the cost of an individual HSCT together with the post-transplant care is approximately USD 320,000, the cost for the annual NBS program plus curative treatment for lymphopenias in Medellín would be approximately USD 3,017,845. By contrast, if newborns with a possible T-cell lymphopenia are not screened at birth, they will develop infections requiring prolonged hospitalization and special treatment with an estimated cost of USD 2,000,000 per year. Thus, the total annual health care costs for the 9 calculated affected newborns with lymphopenia in Medellín would amount to USD 18 million, representing a 5.9 fold increase for the system. Moreover, an undiagnosed child suffering from lymphopenia would hopelessly die in the first five years of live, decreasing the statistics for childhood wellbeing in Colombia, a good indicator of country development. Conclusions: Our results will demonstrate that is profitable and cost-effective to adopt NBS programs for T-cell lymphopenias in Medellín despite administrative and logistics restrictions and more importantly, the educational limitations of the health care agencies and medical community, that have not experienced the advantages of disease prevention and promptly management in primary immunodeficiencies. We pretend to expand this initiative for the early diagnosis of other rare diseases and to establish procedures for the early treatment and complications prevention of affected patients by personalized medicine strategies. Conflict of interest: The authors declare that they have no conflict of interest. Keywords: TRECs, newborn screening (NBS) programs, T-cell lymphopenias in Medellín, Severe Combined Immunodeficiency, DiGeorge Syndrome Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Immunodeficiencies Citation: Sánchez IP, Franco JL, Arias AA, Orrego JC, Giraldo ML, Gutierrez Hincapie S, Rojo FD, Velásquez EM, Quevedo A, Condino-Neto A and Trujillo CM (2015). Pilot study for the neonatal screening of congenital T cell lymphopenias. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00127 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 May 2015; Published Online: 14 Sep 2015. * Correspondence: PhD. Isaura P Sánchez, Corporación Universitaria Remington, Medellin, Antioquia, N/A, Colombia, isapisan@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Isaura P Sánchez Jose L Franco Andres A Arias Julio C Orrego Mónica L Giraldo Sebastian Gutierrez Hincapie Frank D Rojo Enrique M Velásquez Augusto Quevedo Antonio Condino-Neto Claudia M Trujillo Google Isaura P Sánchez Jose L Franco Andres A Arias Julio C Orrego Mónica L Giraldo Sebastian Gutierrez Hincapie Frank D Rojo Enrique M Velásquez Augusto Quevedo Antonio Condino-Neto Claudia M Trujillo Google Scholar Isaura P Sánchez Jose L Franco Andres A Arias Julio C Orrego Mónica L Giraldo Sebastian Gutierrez Hincapie Frank D Rojo Enrique M Velásquez Augusto Quevedo Antonio Condino-Neto Claudia M Trujillo PubMed Isaura P Sánchez Jose L Franco Andres A Arias Julio C Orrego Mónica L Giraldo Sebastian Gutierrez Hincapie Frank D Rojo Enrique M Velásquez Augusto Quevedo Antonio Condino-Neto Claudia M Trujillo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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To summarize the genetic characteristics of 671 Chinese pedigrees affected with Duchenne/Becker muscular dystrophy (DMD/BMD). Clinical data of the pedigrees were collected. Multiplex PCR, multiple ligation dependent probe amplification (MLPA), next generation sequencing (NGS), Sanger sequencing and long read sequencing were used to detect the variant of DMD gene in the probands and their mothers, and prenatal diagnosis was provided for high risk pregnant women. Among 178 pedigrees analyzed by multiplex PCR, 44 variants of the DMD gene were detected, with the genetic diagnosis attained in 110 pedigrees. Among 493 pedigrees analyzed by MLPA in combination with NGS or Sanger sequencing, 294 pathogenic/possible pathogenic variants were identified, among which 45 were unreported previously, and the genetic diagnosis attained in 484 pedigrees. Structural variants of the DMD gene were identified in two pedigrees by long-read sequencing. Among 444 probands, 341 have inherited the DMD gene variant from their mothers (76.8%). Among 390 women with a high-risk, 339 have opted to have natural pregnancy and 51 chose preimplantation genetic testing for monogenetic disease (PGT-M). The detection rate of neonatal patients and carriers following natural pregnancy was significantly higher than that for PGT-M. Combined application of MLPA, NGS, Sanger sequencing and long-read sequencing is an effective strategy to detect DMD/BMD. PGT-M can effectively reduce the risk of fetuses. Above finding has expanded the spectrum of DMD gene variants and provided a basis for reproductive intervention for pregnancies with a high risk for DMD/BMD.

D.O.1 A two-tiered approach to newborn screening for Duchenne muscular dystrophy (DMD) using dried blood spots for sequential CK and DNA analysis

This report summarizes the progress made in newborn screening for Duchenne muscular dystrophy (DMD). This subject was discussed fully at a symposium held on September 11-12, 2012, in Bethesda, Maryland, by a group of experts from multiple disciplines. The meeting was triggered by the simultaneous combination of improvements in methods for newborn screening for DMD and greater potential for treatment. On the screening side, a two-tier system of newborn screening was introduced that enabled creatine kinase levels and DMD gene analysis to be done on the same dried blood spots obtained at birth. Treatment improvements included promising results from exon skipping as well as multiple studies showing long-term benefits of glucocorticoids and data indicating that early intervention of both forms of therapy was the most beneficial. Conclusions from this symposium with supportive data could have a significant impact on propelling efforts for approval of newborn screening for DMD.

Dystrophinopathies are the most common forms of muscular dystrophy in childhood, characterize by a progressive muscle weakness. Mutations in the DMD gene result in Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD), inherited in X-linked recessive mode with a frequency of 1 in 3500 live born males. The present retrospective study aims to describe the mutation spectrum of DMD gene observed in 19 boys with muscular dystrophy without any clinical or familial information addressed to our laboratory between 2016 and 2020. Mutation analysis was performed by PCR multiplex using a primer set of Chamberlain and Beggs. DNA was extracted from peripheral blood and PCR products were separated on 1% agarose gel and detected in U.V light after ethidium bromide staining. The DMD gene is the largest human gene (2.4Mb) on Xp2. It consists of 79 exons encoding for dystrophin. Deletions were observed in 60% of patients, especially in the exons 3–7 and 44–55. To discern between the DMD and DMS diseases we can use a clinical approach or immunohistochemistry techniques. However, the protein-based strategy followed by molecular analysis is probably the most efficient way to approach diagnosis and to propose a correct genetic counselling. &#x0D; Keywords: Dystrophinopathies, Polymerase chain reaction, Duchene Muscular Dystrophy, Becker Muscular Dystrophy.

Missed Opportunities for Duchenne Muscular Dystrophy

Colorectal Cancer Genetics Screening in the Community: Are We Ready? Can We Do It?

The phenotype of Becker muscular dystrophy (BMD) is highly variable, and the disease may be underdiagnosed. We searched for new mutations in the DMD gene in a cohort of previously undiagnosed patients who had been referred in the period 1985-1995. All requests for DNA analysis of the DMD gene in probands with suspected BMD were re-evaluated. If the phenotype was compatible with BMD, and no deletions or duplications were detected, DNA samples were screened for small mutations. In 79 of 185 referrals, no mutation was found. Analysis could be performed on 31 DNA samples. Seven different mutations, including 3 novel ones, were found. Long-term clinical follow-up is described. Refining DNA analysis in previously undiagnosed cases can identify mutations in the DMD gene and provide genetic diagnosis of BMD. A delayed diagnosis can still be valuable for the proband or the relatives of BMD patients.