Duchenne muscular dystrophy newborn screening: the first 50,000 newborns screened in Taiwan

Duchenne muscular dystrophy (DMD) is an X-linked muscular degenerative disorder, leading to wheelchair confinement at 8–12 years of age and the associated cardiomyopathy, with an incidence of 1:3,600–9,300 live male births [1]. Despite advances and increased availability of genetic testing for DMD, the mean age at diagnosis is 3.5–5 years, usually up to 2 years later than the appearance of first symptoms. New treatments for DMD are also developing, including reading-through [2], exon-skipping, and gene therapy [3].

Newborn screening (NBS) for DMD by measuring creatine kinase (CK) activity has been attempted for a few years; however, a high false-positive rate was reported in all programs; therefore, variable strategies including re-screen and molecular testing have been employed [4‐6]. We have added DMD NBS into our routine NBS program since 2021. Here, we report our screening strategy and results of the first year of universal, population-based screening in 50,000 newborns.

The NBS Center at the National Taiwan University Hospital (NTUH) conducts routine newborn screening for approximately 35% born babies around Taiwan. DBS samples were obtained at 48–72 h after birth. DBS CK-MM was measured using a GSP^® Neonatal Creatine Kinase-MM kit with a fully automated GSP instrument (both manufactured by PerkinElmer, Turku, Finland) according to the manufacturer’s instruction. Newborns who had a first-screen CK-MM level higher than cutoffs (the 99^th percentile of normal newborns, 750 ng/mL whole blood (WB) in full term babies and 650 ng/mL in premature babies) were requested to have a re-screen 2 weeks later. The cutoff for re-screen was 300 ng/mL. For those with an elevated CK-MM level at re-screen, defined as persistent hyperCKemia, a 3^rd CK-MM measurement and molecular analysis were requested. The molecular analysis was performed on DNA extracted from DBS using a whole-exome sequencing (WES) muscle panel for the targeted DMD gene and other neuromuscular related genes.

Between Feb. 2021 and Dec. 2021, 50,572 newborns were screened with the CK-MM assay at a mean age of 3 days (SD 3 days). In total, 1.2% (632) of the newborns displayed an elevation in CK-MM in their first samples collected at a mean age of 2 days (SD 1 day), slightly earlier than the population’s practice. Among them, 510 (81%) re-call samples were tested at the mean age of 12 days (SD 8 days) and were returned to normal CK-MM levels. Five subjects (1%) had died of unrelated reasons and were not be able to have another check. Four subjects (one female and three males) were unable to do the re-screen, and WES using the first-screen DBS revealed a variant on COL12A1 gene in a male subject. Fourteen newborns (2%) met the criteria of persistent hyperCKemia, but eleven (7 males and 4 females) of the 14 newborns had normal CK-MM levels in their 3^rd samples. Molecular tests in the remaining 3 subjects with persistent hyperCKemia confirmed as having variants on DMD gene (Fig. 1) in all of them. The first CK-MM values for cases 1, 2, and 3 were 5247, 15983, and 15104 ng/mL, respectively. The 2^nd CK-MM values were 1885, 3058, and 4403 ng/mL, respectively. Finally, the serum CK levels as the confirmatory tests were 4070, 5337, and 3457 U/L, respectively.

We hypothesized that first-screen CK-MM levels could help make the clinical judgement of babies with acid alpha glucosidase (GAA) deficiency or Pompe disease. GAA deficiency was defined as lymphocyte GAA activity <3% of the normal mean and GAA variants on both the maternally and paternally inherited chromosomes; infantile-onset Pompe disease (IOPD) refers to patients with cardiac involvement at the time of diagnosis, and all other cases are classified as later-onset Pompe disease and were followed for symptoms as previously reported [7]. In this period, we encountered 13 subjects with positive Pompe NBS, with 5 GAA deficiency and 8 partial GAA deficiency. Among them, only 1 newborn with CK-MM at 470 ng/mL (Fig. 1 and 2), approximately at the 95^th percentile of the newborn range, had the lowest GAA activity in DBS, and was confirmed as a patient with classical IOPD due to the presence of hypertrophic cardiomyopathy. We have initiated the disease-specific treatment at the age of 9 days. The 2^nd highest CK-MM, 375 ng/mL, was approximately at the 90^th percentile and was found in a newborn diagnosed with later-onset Pompe disease. The other 3 newborns with later-onset Pompe disease and the other 8 newborns with partial GAA deficiency had normal CK-MM levels. None of the 4 subjects with later-onset Pompe disease has been on treatment yet. During this period, we did not encounter newborns who were positive for spinal muscular atrophy (SMA) screening; therefore, there was no information about the utility of CK-MM assay in SMA NBS.

Here we reported a universal NBS for DMD using a FDA-approved immunoassay. In contrast to a re-screen rate of 0.9% (237 out of the 26,135 newborns) in the New York State (NYS) Newborn Screening Program [8], we experienced a high intended re-screen rate (1.2%), in order not to have false negatives as in the Wales program [9] or miss boys with a Becker muscular dystrophy phenotype [4, 5]. Most subjects have normal CK-MM levels at the second screen at 2 weeks of age, because the CK-MM levels indeed stabilized at 1 weeks of life [8]. We could improve the specificity of DMD NBS by adding a 2^nd-tier WES test when the original samples revealed high CK-MM [5, 10]; this will be especially benefit to those who were not able to have a 2^nd sample, say, in the period of COVID-19 pandemic as in our current report, and to whom had excessive stress facing the potential false-positive results. However, the add-on cost was not acceptable in our current program and was not executed yet.

In the current report, we identified newborns with DMD in one out of 8,710 live male newborns. Recent advances such as the increased disease awareness, uptake of expanded carrier screening, and the usage of advanced prenatal testing all should have an impact to the nature occur incidence of DMD. Nevertheless, one recent genotype study in Taiwan showed that only 50% of DMD cases could be detected by multiplex ligation-dependent probe amplification (MLPA) [11], implying the limitation of current carrier or prenatal DMD screening. NBS using a biochemical marker will be necessary for early diagnosis of DMD.

DMD NBS process additional utilities as demonstrated in our report. Since other diseases with the associated CK-MM elevations may be revealed by this, it is useful to conjunction DBS CK-MM to other screening such as Pompe NBS to help the identification of IOPD so that treatment can be start immediately. Only patients with IOPD, but not LOPD, will have the elevation in CK-MM at the first screening sample.

A clear guideline for prospectively followed asymptomatic DMD patients on when and how to initiate treatment is not available yet. Most of the clinical trials for new treatment were conducted in patients aged 4 years or older, even though patients may present with gross motor delay from 4 to 4 months of age [12]. For the use of glucocorticosteroids, only Deflazacort has been approved for patients aged 2 years and older. Long-term outcome data generated from newborn screening will certainly be critical to make a guideline for pre-symptomatic follow-up of DMD.