Ryosuke Bo

Proximal urea-cycle disorders (PUCDs), including N-acetylglutamate synthase deficiency (NAGSD), ornithine transcarbamylase deficiency (OTCD), and carbamoyl phosphate synthase 1 deficiency (CPS1D), cause hyperammonemia and impair neurological outcomes. Early detection of late-onset forms allows presymptomatic intervention to prevent hyperammonemia; however, reliable newborn screening (NBS) markers are lacking. This prospective pilot study in Hyogo Prefecture, Japan, evaluated hypocitrullinemia as a screening marker for late-onset PUCDs. Newborns with citrulline levels below the 0.05th percentile on NBS between June 2020 and May 2024 were enrolled in the study. Confirmatory diagnosis of PUCDs was performed using plasma amino acids, urinary organic acids, and genetic testing. During the first period (101,172 newborns), 11 newborns exhibited hypocitrullinemia; 10 underwent further evaluation. One newborn was diagnosed with CPS1D (compound heterozygous CPS1 variants); another was later diagnosed with Leigh syndrome. The remaining eight cases were false positives, often associated with prematurity, poor feeding, or gastrointestinal disorders. A second dried blood spot (DBS) card protocol was introduced in the second period (34,694 newborns), reducing false positives. One neonatal-onset OTCD case was detected, and citrulline levels were normalized in six of the seven other cases. In summary, hypocitrullinemia can identify presymptomatic PUCDs, and requesting a second DBS card reduces false positives, supporting its feasibility for incorporation into NBS programs.

Background: SMN1 and SMN2 are causative and modifier genes, respectively, for spinal muscular atrophy (SMA). The incidence of SMN1 homozygous deletion in Japan is 1 in 20,000. However, the incidence of SMN2 homozygous deletion in Japan remains unknown. Methods: To clarify the incidence of homozygous SMN2 deletion in Japan, real-time polymerase chain reaction (PCR) was performed on dried blood spot (DBS) samples collected from newborns nationwide. Samples with positive or ambiguous results were retested using PCR-restriction fragment length polymorphism (PCR-RFLP) and nucleotide sequence analysis. Results: Of the 1000 DBS samples that were screened using real-time PCR, 51 were positive. Retesting using PCR-RFLP analysis identified 10 false results: six false positives and four false negatives. Therefore, there were 49 true positives among the 1000 samples. Notably, nucleotide sequence analysis revealed that the false negatives were caused by the cross-reactivity of SMN2 primers with SMN1 sequences. Conclusions: The incidence of homozygous SMN2 deletion in Japan is approximately 1 in 20 people. This incidence is much higher than that of homozygous SMN1 deletion and may reflect the vulnerability of the SMN2 region. Importantly, the results of the present study suggest that false negatives in the screening process were caused by cross-reactivity with non-target gene sequences.

Background/Objectives: Duchenne muscular dystrophy (DMD) is an X-linked inherited muscle disease. Patients with DMD demonstrate improved prognosis with angiotensin-converting enzyme inhibitors and beta-blockers at the time of cardiac dysfunction. However, most deaths due to DMD are due to cardiac dysfunction. Fragmented QRS (fQRS) is an abnormal finding that forms a notch in the QRS wave on electrocardiography (ECG) and is associated with fibrosis and scarring of the myocardium. Methods: Patients with DMD were examined for the number of leads of fQRS, their sites of appearance, changes in cardiac dysfunction, and age using the chest leads of a synthesized 18-ECG. A retrospective analysis of 184 patients under 20 years of age with DMD and known genetic mutations was performed; they were divided into three age groups: 3–10, 11–15, and 16–20 years. The chest leads of the ECG were defined as follows: V1-3, anterior leads; V4-6, lateral leads; V7-9, posterior leads; and V3R-V5R, right-sided chest leads. Cardiac dysfunction was defined as a left ventricular (LV) ejection fraction p = 0.003). The group with LV dilation had a significantly greater number of fQRS leads than did the non-dilation group (p = 0.009). Conclusions: The fQRS site is associated with age, cardiac dysfunction, and LV dilation. Multivariate regression analysis revealed that the number of anterior leads of the fQRS correlated with age and that of lateral leads of the fQRS with cardiac dysfunction and LV dilation. The number of fQRS leads on the lateral wall marks cardiac dysfunction and LV dilation.

Background/Objectives: Nusinersen is a disease-modifying drug for spinal muscular atrophy (SMA) that improves motor function. However, its effects on the skeletal muscles remain unclear. This study aimed to assess the intramuscular fat fraction in patients with SMA types II and III using muscle magnetic resonance imaging (MRI) and to explore the relationship between muscle tissue, lipid metabolism, and motor function during nusinersen treatment. Methods: This study included seven pediatric patients with SMA types II and III who received nusinersen treatment. Muscle MRIs were performed at three time points. Images of the central thigh were used to measure the cross-sectional area (CSA) and muscle fat area, and the intramuscular fat fraction (IMFF) was calculated. The thigh muscles were categorized into three groups: quadriceps, adductor, and hamstrings. Results: The median (range) of total IMFF for SMA type II and III at T-0, T-2, and T-4 were 18.5 (12.6–48.4), 24.4 (10.1–61.4), and 39.0 (30.0–68.6) % and increased over time. In five patients whose motor function was evaluated, a moderate negative correlation was observed between the changes in the Hammersmith Functional Motor Score Expanded (HSFME) and IMFF (r = −0.51). No significant changes in serum triglyceride or total cholesterol levels were observed during treatment. Conclusions: An increase in IMFF was associated with a decline in motor function. The baseline IMFF score was related to improvements in motor function scores, suggesting that the IMFF of the thigh muscle may serve as a novel, objective, and quantitative skeletal muscle-related biomarker for predicting the effects of nusinersen on muscle tissue.

Newborn screening (NBS) for spinal muscular atrophy (SMA) is necessary, as favorable outcomes can be achieved by treatment with disease-modifying drugs in early infancy. Although SMA-NBS has been initiated in Japan, its clinical results have not been fully reported. We report the findings of the initial 2.5 years of a pilot SMA-NBS of approximately 16,000 infants conducted from February 2021 in Hyogo Prefecture, Japan. Clinical data of 17 infants who tested positive were retrospectively obtained from the NBS follow-up centers participating in this multicenter cohort observational study. Genetic testing revealed 14 false positives, and three infants were diagnosed with SMA. Case 1 had two copies of survival motor neuron (SMN) 2 and showed SMA-related symptoms at diagnosis. Case 2 was asymptomatic, with two copies of SMN2. Asymptomatic case 3 had four copies of SMN2 exon 7, including the SMN1/2 hybrid gene. Cases 1 and 2 were treated within 1 month and case 3 at 8 months. All the patients showed improved motor function scores and did not require respiratory support. The identification of infants with SMA via NBS and early treatment improved their motor and respiratory outcomes. Thus, implementation of SMA-NBS at a nationwide scale should be considered.

The survival motor neuron 2 (SMN2) gene is a recognized modifier gene of spinal muscular atrophy (SMA). However, our knowledge about the role of SMN2—other than its modification of SMA phenotypes—is very limited. Discussions regarding the relationship between homozygous SMN2 deletion and motor neuron diseases, including amyotrophic lateral sclerosis, have been mainly based on retrospective epidemiological studies of the diseases, and the precise relationship remains inconclusive. In the present study, we first estimated that the frequency of homozygous SMN2 deletion was ~1 in 20 in Japan. We then established a real-time polymerase chain reaction (PCR)-based screening method using residual dried blood spots to identify infants with homozygous SMN2 deletion. This method can be applied to a future prospective cohort study to clarify the relationship between homozygous SMN2 deletion and motor neuron diseases. In our real-time PCR experiment, both PCR (low annealing temperatures) and blood (high hematocrit values and low white blood cell counts) conditions were associated with incorrect results (i.e., false negatives and positives). Together, our findings not only help to elucidate the role of SMN2, but also aid in our understanding of the pitfalls of current SMA newborn screening programs for detecting homozygous SMN1 deletions.

The authors wish to make the following correction to this paper