Multiplex Real-Time PCR-Based Newborn Screening for Severe Primary Immunodeficiency and Spinal Muscular Atrophy in Osaka, Japan: Our Results after 3 Years.

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases.

Newborn screening for spinal muscular atrophy in Osaka -challenges in a Japanese pilot study.

OBJECTIVE: This study aimed to establish an optional newborn screening program for spinal muscular atrophy (SMA-NBS) in Osaka. METHODS: A multiplex TaqMan real-time quantitative polymerase chain reaction assay was used to screen for SMA. Dried blood spot samples obtained for the optional NBS program for severe combined immunodeficiency, which covers about 50% of the newborns in Osaka, were used. To obtain informed consent, participating obstetricians provided information about the optional NBS program to all parents by giving leaflets to prospective parents and uploading the information onto the internet. We prepared a workflow so that babies that were diagnosed with SMA through the NBS could be treated immediately. RESULTS: From 1 February 2021 to 30 September 2021, 22,951 newborns were screened for SMA. All of them tested negative for survival motor neuron (SMN)1 deletion, and there were no false-positives. Based on these results, an SMA-NBS program was established in Osaka and included in the optional NBS programs run in Osaka from 1 October 2021. A positive baby was found by screening, diagnosed with SMA (the baby possessed 3 copies of the SMN2 gene and was pre-symptomatic), and treated immediately. CONCLUSION: The workflow of the Osaka SMA-NBS program was confirmed to be useful for babies with SMA.

Stability and Oligomerization of Mutated SMN Protein Determine Clinical Severity of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. Approximately 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) gene deletion, while ~5% carry an intragenic SMN1 mutation. Here, we investigated the stability and oligomerization ability of mutated SMN1 proteins. Plasmids containing wild- and mutant-type SMN1 cDNA were constructed and transfected into HeLa cells. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar abundances of transcripts from the plasmids containing SMN cDNA, but Western blotting showed different expression levels of mutated SMN1 proteins, reflecting the degree of their instability. A mutated SMN1 protein with T274YfsX32 exhibited a much lower expression level than other mutated SMN1 proteins with E134K, Y276H, or Y277C. In immunoprecipitation analysis, the mutated SMN1 protein with T274YfsX32 did not bind to endogenous SMN1 protein in HeLa cells, suggesting that this mutation completely blocks the oligomerization with full-length SMN2 protein in the patient. The patient with T274YfsX32 showed a much more severe phenotype than the other patients with different mutations. In conclusion, the stability and oligomerization ability of mutated SMN1 protein may determine the protein stability and may be associated with the clinical severity of SMA caused by intragenic SMN1 mutation.

Spinal Muscular Atrophy: Diagnosis, Incidence, and Newborn Screening in Japan.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder that causes degeneration of anterior horn cells in the human spinal cord and subsequent loss of motor neurons. The severe form of SMA is among the genetic diseases with the highest infant mortality. Although SMA has been considered incurable, newly developed drugs-nusinersen and onasemnogene abeparvovec-improve the life prognoses and motor functions of affected infants. To maximize the efficacy of these drugs, treatments should be started at the pre-symptomatic stage of SMA. Thus, newborn screening for SMA is now strongly recommended. Herein, we provide some data based on our experience of SMA diagnosis by genetic testing in Japan. A total of 515 patients suspected of having SMA or another lower motor neuron disease were tested. Among these patients, 228 were diagnosed as having SMA with survival motor neuron 1 (SMN1) deletion. We analyzed the distribution of clinical subtypes and ages at genetic testing in the SMN1-deleted patients, and estimated the SMA incidence based on data from Osaka and Hyogo prefectures, Japan. Our data showed that confirmed diagnosis by genetic testing was notably delayed, and the estimated incidence was 1 in 30,000-40,000 live births, which seemed notably lower than in other countries. These findings suggest that many diagnosis-delayed or undiagnosed cases may be present in Japan. To prevent this, newborn screening programs for SMA (SMA-NBS) need to be implemented in all Japanese prefectures. In this article, we also introduce our pilot study for SMA-NBS in Osaka Prefecture.

Epilepsy in patients with advanced Fukuyama congenital muscular dystrophy.

BACKGROUND: Recent advances in respiratory management have improved survival for patients with Fukuyama congenital muscular dystrophy (FCMD), characterized by congenital muscular dystrophy and brain malformation. Previous studies reported that more than half of patients exhibit seizures in childhood. However, little is known about epilepsy after childhood. METHODS: To elucidate the long-term clinical course of epilepsy, we retrospectively reviewed all medical records in nine patients (6 males, mean age 20.7 years) with FCMD diagnosed between 1981 and 2019. RESULTS: The follow-up periods ranged from 6 to 30 years (mean 18.4 years). A total of 75 EEG recordings were available from nine patients. In some patients, EEGs were normal during early childhood but tended to show paroxysmal discharges with age. Overall, epileptic seizures were observed in six patients. Except for one presenting with afebrile seizure at one year of age, the remaining five patients developed epilepsy between 13 and 22 years of age. The most common seizure type was focal impaired awareness seizure. After adolescence, four patients exhibited status epilepticus. Their convulsive movements of the seizures became less prominent with progression of the disease. At the last evaluation, most patients (5/6) had uncontrolled seizures. CONCLUSIONS: Despite presence of distinct brain malformation, epileptic seizures may develop after childhood in FCMD patients. Our experience suggests that clinicians should be careful not to overlook epileptic seizures, especially in advanced-stage patients who had profound muscle weakness.

Methylprednisolone pulse therapy in 31 patients with refractory epilepsy: A single-center retrospective analysis.

PURPOSE: We investigated the efficacy of methylprednisolone pulse therapy (MP) and responder characteristics in patients with refractory epilepsy. METHODS: We reviewed medical records of our center to identify patients with refractory epilepsy treated with MP other than continuous spikes and waves during slow sleep (CSWS), Landau-Kleffner syndrome (LKS), or Rasmussen's syndrome (RS) between 2004 and 2015. A course of MP consisted of intravenous methylprednisolone (30 mg/kg/day) on three consecutive days. Patients received multiple courses at intervals of four weeks. We examined seizure outcome, developmental outcome, antibodies to N-methyl-d-aspartate (NMDA)-type glutamate receptors (GluRs), cerebral spinal fluid (CSF)-albumin/serum-albumin ratio, and interictal electroencephalograms (EEGs). Responder to MP was defined as maintaining seizure reduction rate (SRR) ≥50% for three months after the first course of MP. RESULTS: Thirty-one consecutive patients treated with MP at our center were studied. Seizure types were focal onset impaired awareness seizure (FIAS) only (n = 23), FIAS with epileptic spasms (ES) (n = 7), and ES only (n = 1). Responder rate was 32.2% (10/31 patients), and seizure-free rate was 9.7% (3/31). Responders constituted 43.5% of patients without ES. No patient with ES was responder. Behavior and cognition also improved in 6 of 10 responders. History of seizure aggravation after inactivated vaccine before MP was found significantly higher rate in responder patients, comparing with nonresponder patients (p = 0.01). CONCLUSION: Methylprednisolone pulse therapy may be considered for possible treatment in patients with focal epilepsy with drug-resistant seizures without ES, and it may improve cognitive function and behavioral comorbidities.

Comprehensive analysis of coding variants highlights genetic complexity in developmental and epileptic encephalopathy.

Although there are many known Mendelian genes linked to epileptic or developmental and epileptic encephalopathy (EE/DEE), its genetic architecture is not fully explained. Here, we address this incompleteness by analyzing exomes of 743 EE/DEE cases and 2366 controls. We observe that damaging ultra-rare variants (dURVs) unique to an individual are significantly overrepresented in EE/DEE, both in known EE/DEE genes and the other non-EE/DEE genes. Importantly, enrichment of dURVs in non-EE/DEE genes is significant, even in the subset of cases with diagnostic dURVs (P = 0.000215), suggesting oligogenic contribution of non-EE/DEE gene dURVs. Gene-based analysis identifies exome-wide significant (P = 2.04 × 10-6) enrichment of damaging de novo mutations in NF1, a gene primarily linked to neurofibromatosis, in infantile spasm. Together with accumulating evidence for roles of oligogenic or modifier variants in severe neurodevelopmental disorders, our results highlight genetic complexity in EE/DEE, and indicate that EE/DEE is not an aggregate of simple Mendelian disorders.

Chronic dysfunction of blood-brain barrier in patients with post-encephalitic/encephalopathic epilepsy.

PURPOSE: This study aimed to elucidate the characteristics and effects of chronic blood-brain barrier (BBB) dysfunction in patients with post-encephalitic/encephalopathic epilepsy (PEE), using brain images and the cerebral spinal fluid (CSF)/serum albumin ratio (albumin quotient, QAlb) as a marker of BBB function. METHODS: We examined the albumin levels in CSF and serum samples from 312 patients with refractory epilepsy in our center between 2004 and 2015. Sixty samples from patients with PEE and 97 samples from age- and sex-matched disease controls (DC) were evaluated. We classified PEE patients into a widespread lesion group and a focal lesion group by severity on brain magnetic resonance images in the chronic phase after acute encephalitis/encephalopathy. RESULTS: Median QAlb was higher in PEE than in DC [median (range) ×103: PEE 3.6 (1.0-10.3) versus DC 2.7 (1.0-6.7), p = 0.007]. In a linear regression analysis of the relationship between QAlb and patient's age at CSF examination or duration of epilepsy, the slope of the regression line was greater in PEE than in DC. Furthermore, in patients under ten years of age, linear regression analysis of QAlb versus seizure frequency showed a weak but positive correlation. Among PEE patients, seizure frequency was higher in the widespread lesion group than in the focal lesion group [300 (4-3000) versus 30 (1-1500) seizures/month, p < 0.001]. CONCLUSION: Our study suggests that patients with PEE have more severe BBB dysfunction, and that the BBB dysfunction is associated with refractory epilepsy.

A case of early onset epileptic encephalopathy with de novo mutation in SLC35A2: Clinical features and treatment for epilepsy.

INTRODUCTION: Mutations of SLC35A2 that encodes Golgi-localized Uridine diphosphate (UDP)-galactose transporter at Xp11.23 lead to congenital disorders of glycosylation (CDG). Although patients with CDG generally have diverse systemic symptoms, patients with a SLC35A2 mutation manifest predominantly disorders of the central nervous system (CNS). CASE REPORT: A female infant aged 12months was referred to our center because of intractable seizures. The patient was born with birth weight of 3228g after 40weeks of unremarkable gestation. At the age of 2months, she had partial seizures evolving to epileptic spasms. Her electroencephalogram showed hypsarrhythmia. Her seizures were refractory to antiepileptic drugs. At referral to our center at 12months, she had developmental delay (no head control), widely spaced inverted nipples, external strabismus, and bilateral heterochromia of irises. Blood examinations were normal. Brain magnetic resonance imaging findings included cerebral and cerebellar atrophy, thinning of the corpus callosum, and arachnoid pouch. Whole-exome sequencing detected a de novo frameshift mutation c.950delG (p.Gly317Alafs*32) at exon 4 in SLC35A2. Seizures subsided after the second adrenocorticotropic hormones (ACTH) therapy at 18months. At the age of 36months, although she had intellectual disability with no meaningful words, she was seizure-free and was able to sit without support and showed smiling face a lot. CONCLUSION: This report reviewed the clinical features of patients with a SLC35A2 mutation. ACTH therapy may be effective for refractory epilepsy in these patients.

Benign neonatal sleep myoclonus: our experience of 15 Japanese cases.

PURPOSE: Benign neonatal sleep myoclonus is a non-epileptic movement disorder that may mimic neonatal seizures. The aim of this study was to clarify the clinical manifestations and outcomes in Japanese infants with benign neonatal sleep myoclonus. METHODS: We reviewed the clinical manifestations and outcomes in 15 consecutive patients with benign neonatal sleep myoclonus (males: 10), including three paired familial cases, referred to our center between 1996 and 2011. The diagnosis of benign neonatal sleep myoclonus was based on a neonatal onset, characteristic myoclonic jerks that occurred during sleep, and normal electroencephalogram findings. RESULTS: All were healthy full-term neonates at birth. The age at onset ranged from 1 to 18 days (median: 7 days). Prior to referral to our center (3-8 weeks), two infants had been placed on antiepileptic drugs, without effects. During the clinical course, the myoclonic jerks resolved by 6 months in 14 of the 15 patients. On follow-up (final evaluation, mean: 38 months), all but one patient (speech delay) showed normal development. None developed epilepsy. Of note, migraine occurred after 5 years of age in three children, including one who developed cyclic vomiting syndrome, evolving to migraine. Another boy developed cyclic vomiting syndrome, a precursor of migraine, before 1 year, and was being followed. A high incidence of migraine was observed in five (42%) of 12 parents whose detailed family history was available. CONCLUSION: Our study suggests that benign neonatal sleep myoclonus is related to migraine. With the high rate of familial cases, further genetic study, including migraine-related gene analysis, is necessary to determine the underlying mechanism responsible for benign neonatal sleep myoclonus.

Ictal 99mTc-Ethyl Cysteinate Dimer SPECT Findings of a Girl With Refractory Localization-Related Epilepsy Who Developed Transient Ictal Bradycardia.

Ictal bradycardia, which is considered to be one of the causes of sudden unexplained death in epilepsy, is rare. A 10-year-old girl with focal cortical dysplasia in her right centroparietal region developed transient ictal bradycardia during cluster seizures. Brain magnetic resonance imaging demonstrated a high signal intensity lesion adjacent to the focal cortical dysplasia lesion. Ictal 99mTc-ethyl cysteinate dimer single-photon emission computed tomography (SPECT) detected hyperperfusion in an area containing the high signal intensity lesion, which was located close to the insular cortex. Since the hyperperfusion zone observed on SPECT was considered to reflect seizure propagation, it is possible that the ictal bradycardia experienced in the present case was caused by the following mechanism: The repetitive seizure activity caused the high-intensity lesion seen on MRI to expand into the right insular cortex, which controls cardiac rhythm, resulting in ictal bradycardia.

[Successful treatment with intravenous steroid pulse therapy of a boy with recurrent idiopathic sixth nerve palsy].

A 3-year-old boy developed left-sided convergent strabismus one week after upper respiratory infection. All examinations, including analysis of cerebrospinal fluid, a tensilon test, and brain MRI, were negative. He was diagnosed with idiopathic sixth nerve palsy. His symptom resolved gradually with vitamin B12, and remitted completely three months after onset. At the age of 6 years, he experienced recurrence of left-sided sixth nerve palsy. After vitamin B12 failed, his symptom responded markedly to intravenous steroid pulse therapy starting on day 26 after relapse. He has been symptom-free for three years since the second remission. Steroid therapy might be effective, and should be considered in children with idiopathic sixth nerve palsy who do not show spontaneous remission.

Predictive value of EEG findings at control of epileptic spasms for seizure relapse in patients with West syndrome.

PURPOSE: To evaluate the prognostic importance of electroencephalography (EEG) findings at cessation of epileptic spasms for seizure outcome. METHODS: We reviewed 71 children with West syndrome (cryptogenic 14) who had obtained control of epileptic spasms with initial treatment (adrenocorticotropic hormone (ACTH) 37, high-dose vitamin B6 2, and antiepileptic drugs 32). According to the EEG findings at control of epileptic spasms, the subjects were divided into three groups: normal group (no epileptic activity, n=12), abnormal group (residual epileptic activity without hypsarrhythmia, n=53), and hypsarrhythmic group (persisting hypsarrhythmia, n=6). RESULTS: Overall, 47 (66%) of the 71 patients (cryptogenic 4) had experienced relapses of seizures (epileptic spasms 23 and focal seizure 24) after initial control of epileptic spasms. Within symptomatic cases, seizure relapse rate varied widely from 0% (Down syndrome) to 100% (tuberous sclerosis), depending on underlying causes. Seizure relapse depended on the EEG findings at control of epileptic spasms. The normal group had a significantly lower seizure relapse rate (17%) in comparison with the abnormal group (75%), the hypsarrhythmic group (83%), and the epileptiform (abnormal plus hypsarrhythmic, 76%) group. No significant difference in seizure relapse rate was observed between non-hypsarrhythmic (normal plus abnormal, 65%) and hypsarrhythmic groups. At the last follow-up, normal group children also showed a favorable seizure prognosis (seizure control 100%). CONCLUSIONS: A favorable seizure prognosis is associated with the disappearance of epileptic activity, but not the resolution of hypsarrhythmic pattern on EEG at control of epileptic spasms. We suggest that effective treatment for West syndrome should produce both cessation of epileptic spasms and disappearance of epileptic activity on EEG.

[Evaluation of six cases with hypothalamic hamartoma: the relationship between MRI findings and clinical features].

OBJECTIVE: We evaluated the relationship between MRI findings and clinical features in patients with hypothalamic hamartoma (HH). METHODS: We retrospectively reviewed MRI and clinical data (mental retardation, precocious puberty, behavioral problems, and epilepsy) in six patients (3 males and 3 females, ages 12 to 26) with HH. Based on the MRI classification by Arita, HH was classified into two types: parahypothalamic (P) and intrahypothalamic (I). RESULTS: Only one patient was classified as having P-type HH and five were classified as having I-type HH. The patient with P-type HH (diameter 21 mm) showed precocious puberty and mild behavioral problems, but did not developed epilepsy. On the other hand, all patients with I-type HH (diameter 10-32 mm, median 17 mm) developed epilepsy and behavioral problems. Except for one patient, who had the smallest sized HH, I-type four patients developed mental retardation and precocious puberty. Among patients with I-type HH, the size of the tumor was inversely correlated with the age at epilepsy onset and with the degree of mental retardation (DQ/IQ). CONCLUSION: Our data suggested that the MRI classification by Arita, when combined with tumor size, might be helpful in predicting the clinical manifestations in patients with HH.

[Three cases of human herpesvirus-6 encephalopathy showing hyperperfusion in the acute phase on SPECT].

Brain hypoperfusion observed on single-photon emission computed tomography (SPECT) is a typical finding in the acute phase of human herpesvirus-6 (HHV-6) encephalopathy. However, from 2004 to 2010, we encountered three cases of HHV-6 encephalopathy in which hyperperfusion in the area of the brain lesion was observed on SPECT performed within 48 hours after disease onset. The hyperperfusion in the brain was followed by hypoperfusion in the recovery phase. These cases suggest that hyperperfusion may appear in damaged areas prior to the hypoperfusion that is normally associated with HHV-6 encephalopathy.