Uncovering hidden genetic variations: long-read sequencing reveals new insights into tuberous sclerosis complex.

Background: Tuberous sclerosis is a multi-system disorder caused by mutations in either TSC1 or TSC2. The majority of affected patients (85%-90%) have heterozygous variants, and a smaller number (around 5%) have mosaic variants. Despite using various techniques, some patients still have "no mutation identified" (NMI). Methods: We hypothesized that the causal variants of patients with NMI may be structural variants or deep intronic variants. To investigate this, we sequenced the DNA of 26 tuberous sclerosis patients with NMI using targeted long-read sequencing. Results: We identified likely pathogenic/pathogenic variants in 13 of the cases, of which 6 were large deletions, four were InDels, two were deep intronic variants, one had retrotransposon insertion in either TSC1 or TSC2, and one was complex rearrangement. Furthermore, there was a de novo Alu element insertion with a high suspicion of pathogenicity that was classified as a variant of unknown significance. Conclusion: Our findings expand the current knowledge of known pathogenic variants related to tuberous sclerosis, particularly uncovering mosaic complex structural variations and retrotransposon insertions that have not been previously reported in tuberous sclerosis. Our findings suggest a higher prevalence of mosaicism among tuberous sclerosis patients than previously recognized. Our results indicate that long-read sequencing is a valuable approach for tuberous sclerosis cases with no mutation identified (NMI).

Exploring Infantile Epileptic Spasm Syndrome: A Proteomic Analysis of Plasma Using the Data-Independent Acquisition Approach.

This study aimed to identify characteristic proteins in infantile epileptic spasm syndrome (IESS) patients' plasma, offering insights into potential early diagnostic biomarkers and its underlying causes. Plasma samples were gathered from 60 patients with IESS and 40 healthy controls. Data-independent acquisition proteomic analysis was utilized to identify differentially expressed proteins (DEPs). These DEPs underwent functional annotation through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Gene set enrichment analysis (GSEA) was employed for both GO (GSEA-GO) and KEGG (GSEA-KEGG) analyses to examine the gene expression profiles. Receiver operating characteristic (ROC) curves assessed biomarkers' discriminatory capacity. A total of 124 DEPs were identified in IESS patients' plasma, mainly linked to pathways, encompassing chemokines, cytokines, and oxidative detoxification. GSEA-GO and GSEA-KEGG analyses indicated significant enrichment of genes associated with cell migration, focal adhesion, and phagosome pathways. ROC curve analysis demonstrated that the combination of PRSS1 and ACTB, PRSS3, ACTB, and PRSS1 alone exhibited AUC values exceeding 0.7. This study elucidated the significant contribution of cytokines, chemokines, oxidative detoxification, and phagosomes to the IESS pathogenesis. The combination of PRSS1 and ACTB holds promise as biomarkers for the early diagnosis of IESS.

White-Sutton syndrome and congenital heart disease: case report and literature review.

BACKGROUND: White-Sutton syndrome is an autosomal dominant neurodevelopmental disorder caused by heterozygous mutation in POGZ (Pogo Transposable Element Derived with ZNF Domain). This syndrome is characterized by delayed psychomotor development apparent in infancy and abnormal facial features. To date, 80 cases have been reported in the literature; however, the phenotypic characterizations remain incomplete. CASE PRESENTATION: We herein describe a 2-year-old girl harboring a novel frameshift de novo POGZ variant: c.2746del (p.Thr916ProfsTer12). This patient presented with multisystem abnormalities affecting the digestive tract and neurological functioning, as well as congenital heart disease, which involved an atrial septal defect (18 × 23 × 22 mm) with pulmonary arterial hypertension (42 mmHg). The relationship between congenital heart disease and White-Sutton syndrome as described in both the GeneReview and OMIM databases (#616,364) remains unclear. A review of the current literature revealed 18 cases of White-Sutton syndrome with POGZ variants and congenital heart disease, and we summarize their clinical features in this study. CONCLUSIONS: Our findings based on the present case and those in the literature indicate a relationship between POGZ mutation and congenital heart disease.

Identification of a Novel Canonical Splice Site Variant TSC2 c.2967-1G>T That is Not Associated With Tuberous Sclerosis Pathogenesis.

Tuberous sclerosis, also known as tuberous sclerosis complex (TSC), is an autosomal dominant defect characterized by hamartomas in multiple organ systems. Inactivating variants cause this defect in either the TSC1 gene or the TSC2 gene, leading to hamartin or tuberin protein dysfunction, thus resulting in TSC. The diagnostic criteria for TSC suggest that it can be diagnosed by identifying a heterozygous pathogenic variant of TSC1 or TSC2, even in the absence of clinical signs. In a 4-year-old girl, we identified a splicing variant (NM_000548.4: c.2967-1G>T) that she inherited from her father. Neither the girl (patient) nor her father showed typical features of TSC. This variant is located in a NAGNAG acceptor, which can produce mRNA isoforms that differ by a three-nucleotide indel. Reverse transcription polymerase chain reaction analysis of the patient and both parents' blood RNA samples suggested two different splicing patterns, and these two splicing patterns differed in the presence or absence of the first codon of exon 27, thus providing two splicing products designated as isoforms A and B, respectively. Furthermore, the proportions of these two patterns varied between the patient and either parent. A minigene assay further confirmed that the c.2967-1G>T variant led to the absence of isoform A (including the first codon of exon 27). The finding of our study demonstrates this variant, c.2967-1G>T, disrupts the balance of an alternative splice event which involves the use of two tandem alternatives acceptors and is not associated with typical symptoms of tuberous sclerosis. Our finding is of importance for genetic counseling and suggests that we need to be vigilant to avoid misdiagnosis when we encounter such a site.

Non-convulsive Status Epilepticus in SEMA6B-Related Progressive Myoclonic Epilepsy: A Case Report With Literature Review.

Progressive myoclonic epilepsy (PME) is a group of rare diseases characterized by progressive myoclonus, cognitive impairment, ataxia, and other neurologic deficits. PME has high genetic heterogeneity, and more than 40 genes are reportedly associated with this disorder. SEMA6B encodes a member of the semaphorin family and was first reported to cause PME in 2020. Herein, we present a rare case of PME due to a novel SEMA6B gene mutation in a 6-year-old boy born to healthy non-consanguineous Chinese parents. His developmental milestones were delayed, and he developed recurrent atonic seizures and myoclonic seizures without fever at 3 years and 11 months of age. He experienced recurrent myoclonic seizures, non-convulsive status epilepticus (NCSE), atonic seizures, and atypical absence seizures during the last 2 years. At different time points since onset, valproic acid, levetiracetam, piracetam, and clobazam were used to control the intractable seizures. Notably, NCSE was controlled by a combination of piracetam with clobazam and valproic acid instead of intravenous infusion of midazolam and phenobarbital. Due to the limited number of cases reported to date, the clinical description of our case provides a better understanding of the genotype-phenotype correlations associated with PME and indicate that piracetam may be effective against NCSE in patients with SEMA6B-related PME.

A de novo PUM1 Variant in a Girl With a Dravet-Like Syndrome: Case Report and Literature Review.

In the recent 3 years, subjects with Pumilio1-associated developmental disability, ataxia, and seizure syndrome have been identified as harboring Pumilio homolog 1 (PUM1) mutations. However, the characteristics of the seizure phenotype remain to be elucidated. We herein described a 3-year-old female proband who was diagnosed with developmental and epileptic encephalopathy presenting with some features suggestive of a Dravet-like syndrome. For genetic analyses, trio-based whole-exome sequencing and array comparative genomic hybridization were performed. Consequently, a de novo heterozygous missense variant was identified in exon 22 of the PUM1 gene: NM_001020658: c.3439C > T (p.Arg1147Trp). Upon thoroughly reviewing the existing literature, nine cases of PUM1 mutation-related epilepsy were identified, and their clinical features were summarized. A relationship between PUM1 mutation and clinical manifestations characteristic of a Dravet-like syndrome was proposed. To our knowledge, this is the first report of a patient with PUM1 mutation presenting with a Dravet-like syndrome.

Long-term outcomes of infantile spasms in children treated with ketogenic diet therapy in combination with anti-seizure medications in a resource-limited region.

Objective: Despite numerous guidelines, the overall outcome of infantile spasms is poor, with only a small number of patients being able to attend school. The purpose of this study was to investigate long-term outcomes. Patients had poor access to the recommended first-line anti-seizure medications (ASMs), such as hormones (corticotropin or prednisolone/prednisone) and vigabatrin, and their alternative treatment was other ASMs and a ketogenic diet. Methods: Patients suffering from infantile spasms who had at least 2 years of medical records in the electronic medical record system between January 2014 and August 2022 were included in this study. Patient information was retrospectively reviewed. All patients had received ketogenic diet therapy (mainly classical ketogenic diet therapy). The ketogenic diet therapy was combined with ASMs not used as first-line therapies. The primary endpoint outcome measure was the number of patients with seizure freedom. The secondary measures included the duration of ketogenic diet therapy, choice of ASMs, and patient development at the last visit. Results: A total of 177 patients with infantile spasms were included, and 152 (86%) of them had seizure freedom. The median duration from the first to the last hospital visit was 53.27 months, and the number of visits was 47.00. The median age at the initial hospital visit was 8.00 months, and the median age at initiation of the ketogenic diet was 17.73 months. At the last visit, the proportions of patients with neurodevelopmental delay, developmental epileptic encephalopathy, drug-resistant epilepsy, and generalized seizures increased significantly. The frequently used ASMs were topiramate, valproic acid, levetiracetam, nitrazepam, and vitamin B6 injection, while the recommended first-line drugs corticotropin and vigabatrin were rarely selected. The study duration of 9.5 years was divided into three periods but the prescription of ASMs did not change significantly between these periods. Conclusions: Although the seizure freedom rate was high with ketogenic diet therapy combined with non-standard ASMs, the patients had a significant neurodevelopmental delay at the last visit, which was, however, similar to that of standard treatment. To improve the outcomes of infantile spasms, multicenter clinical trials of the ketogenic diet as a first-line treatment in combination with non-standard ASMs are needed.

Pyopneumothorax from coinfection by Trichomonas tenax and Geotrichum capitatum in a child from China: a case report.

BACKGROUND: Trichomonas tenax may appear in the oral cavity of humans due to poor dentition or oral hygiene. Pyopneumothorax is a serious complication of lower respiratory tract infections that very rarely can be caused by a trichomonad species in predisposed individuals. We report a rare case of pleurisy due to T. tenax with coinfection by a fungus. CASE PRESENTATION: We describe a 16-year-old patient with cerebral palsy who presented with severe pyopneumothorax. T. tenax was identified by microscopic examination of the pleural effusion and next-generation sequencing. We also identified Geotrichum capitatum in the pleural effusion and bronchoalveolar lavage fluid cultures. Treatment with voriconazole and metronidazole successfully eliminated these pathogens and relieved the clinical symptoms. A literature review indicated this is the first reported case of pleurisy due to T. tenax with coinfection by a fungus. CONCLUSION: The rarity of pyopneumothorax caused by T. tenax coinfection with a fungus should not be overlooked in the clinic. These patients should be and treated in a timely manner.

Dynamic Changes of Astrocytes and Adenosine Signaling in Rat Hippocampus in Post-status Epilepticus Model of Epileptogenesis.

It is of great importance to explore the development of epileptogenesis, and the adenosine and adenosine kinase (ADK) system seems to play a key role in this process. The aim of this study is to explore the dynamic changes of astrocytes and adenosine signaling during epileptogenesis in rat hippocampus in a post-status epileptogenesis (SE) model. Rat SE models were built and killed for experiments at 1 day (acute phase of epileptogenesis), 5 days (latent phase), 4 weeks (chronic phase), and 8 weeks (late chronic phase of epileptogenesis) after SE induction. Immunofluorescence staining, high-performance liquid chromatography, and Western blotting were performed to assess changes of astrocytes, adenosine, ADK, and ADK receptors (including A1R, A2aR, A2bR, and A3R) in hippocampus. The expression level of glial fibrillary acidic protein significantly increased from latent to late chronic phase. The concentration of adenosine sharply increased in acute phase and gradually decreased in the remaining phases of post-SE, being significantly lower than in the control group in late chronic phase. Protein levels of A1R and A2aR in post-SE models increased in acute phase, whereas A2bR and A3R protein expression decreased in latent phase, chronic phase, and late chronic phase following post-SE epileptogenesis. Protein expression of ADK significantly increased during latent phase, chronic phase, and late chronic phase of post-SE epileptogenesis. In conclusion, the levels of adenosine and protein expression of A1R and A2R significantly increased during acute phase of post-SE. During the remaining phases of post-SE epileptogenesis, there was imbalance among astrocytes, adenosine, adenosine receptors, and ADK. Regulation of the ADK/adenosine system may provide potential treatment strategies for epileptogenesis.

Daily prednisone treatment in Duchenne muscular dystrophy in southwest China.

INTRODUCTION: In this study we aimed to determine the influence of daily prednisone treatment in Duchenne muscular dystrophy (DMD) by performing a prospective, randomized, placebo-controlled trial in southwestern China. METHODS: Sixty-six children with DMD (4-12 years of age) were divided randomly into prednisone and placebo groups. Efficacy and safety of daily prednisone at 0.75 mg/kg/day were evaluated over 12 months by muscle strength and function, quality of life (QoL), quantitative muscle ultrasound (QMUS), and side effects. RESULTS: Significant improvements in muscle strength and function, QoL, and QMUS were observed in the prednisone group compared with the placebo-treated group (P < 0.05). Changes in body weight, height, body mass index, and diastolic blood pressure were similar in both groups (P > 0.05). CONCLUSIONS: This pilot study in southwestern China found that daily prednisone at 0.75 mg/kg/day is suitable for children with DMD. It slowed disease progression and improved QoL and QMUS. Moderate side effects were generally well tolerated.

Serum miR-206 and other muscle-specific microRNAs as non-invasive biomarkers for Duchenne muscular dystrophy.

Creatine kinase has been utilized as a diagnostic marker for Duchenne muscular dystrophy (DMD), but it correlates less well with the DMD pathological progression. In this study, we hypothesized that muscle-specific microRNAs (miR-1, -133, and -206) in serum may be useful for monitoring the DMD pathological progression, and explored the possibility of these miRNAs as potential non-invasive biomarkers for the disease. By using real-time quantitative reverse transcription-polymerase chain reaction in a randomized and controlled trial, we detected that miR-1, -133, and -206 were significantly over-expressed in the serum of 39 children with DMD (up to 3.20 ± 1.20, 2(-ΔΔCt) ): almost 2- to 4-fold enriched in comparison to samples from the healthy controls (less than 1.15 ± 0.34, 2(-ΔΔCt) ). To determine whether these miRNAs were related to the clinical features of children with DMD, we analyzed the associations compared to creatine kinase. There were very good inverse correlations between the levels of these miRNAs, especially miR-206, and functional performances: high levels corresponded to low muscle strength, muscle function, and quality of life. Moreover, by receiver operating characteristic curves analyses, we revealed that these miRNAs, especially miR-206, were able to discriminate DMD from controls. Thus, miR-206 and other muscle-specific miRNAs in serum are useful for monitoring the DMD pathological progression, and hence as potential non-invasive biomarkers for the disease. There has been a long-standing need for reliable, non-invasive biomarkers for Duchenne muscular dystrophy (DMD). We found that the levels of muscle-specific microRNAs, especially miR-206, in the serum of DMD were 2- to 4-fold higher than in the controls. High levels corresponded to low muscle strength, muscle function, and quality of life (QoL). These miRNAs were able to discriminate DMD from controls by receiver operating characteristic (ROC) curves analyses. Thus, miR-206 and other muscle-specific miRNAs are useful as non-invasive biomarkers for DMD.

Altered hippocampal myelinated fiber integrity in a lithium-pilocarpine model of temporal lobe epilepsy: a histopathological and stereological investigation.

The damage of white matter, primarily myelinated fibers, in the central nervous system (CNS) of temporal lobe epilepsy (TLE) patients has been recently reported. However, limited data exist addressing the types of changes that occur to myelinated fibers inside the hippocampus as a result of TLE. The current study was designed to examine this issue in a lithium-pilocarpine rat model. Investigated by electroencephalography (EEG), Gallyas silver staining, immunohistochemistry, western blotting, transmission electron microscopy, and stereological methods, the results showed that hippocampal myelinated fibers of the epilepsy group were degenerated with significantly less myelin basic protein (MBP) expression relative to those of control group rats. Stereological analysis revealed that the total volumes of hippocampal formation, myelinated fibers, and myelin sheaths in the hippocampus of epilepsy group rats were decreased by 20.43%, 49.16%, and 52.60%, respectively. In addition, epilepsy group rats showed significantly greater mean diameters of myelinated fibers and axons, whereas the mean thickness of myelin sheaths was less, especially for small axons with diameters from 0.1 to 0.8µm, compared to control group rats. Finally, the total length of the myelinated fibers in the hippocampus of epilepsy group rats was significantly decreased by 56.92%, compared to that of the control group, with the decreased length most prominent for myelinated fibers with diameters from 0.4 to 0.8µm. This study is the first to provide experimental evidence that the integrity of hippocampal myelinated fibers is negatively affected by inducing epileptic seizures with pilocarpine, which may contribute to the abnormal propagation of epileptic discharge.

Reliability and validity of the Chinese version of the Pediatric Quality Of Life InventoryTM (PedsQLTM) 3.0 neuromuscular module in children with Duchenne muscular dystrophy.

BACKGROUND: The Pediatric Quality of Life Inventory(TM) (PedsQL(TM)) is a widely used instrument to measure pediatric health-related quality of life (HRQOL) in children aged 2 to 18 years. The current study aimed to evaluate the reliability and validity of the Chinese version of the PedsQL(TM) 3.0 Neuromuscular Module in children with Duchenne muscular dystrophy (DMD). METHODS: The PedsQL(TM) 3.0 Neuromuscular Module was translated into Chinese following PedsQL(TM) Measurement Model Translation Methodology. The Chinese version scale was administered to 56 children with DMD and their parents, and the psychometric properties were evaluated. RESULTS: The missing value percentages for each item of the Chinese version scale ranged from 0.00% to 0.54%. Internal consistency reliability approached or exceeded the minimum reliability standard of α = 0.7 (child α = 0.81, parent α = 0.86). Test-retest reliability was satisfactory, with intraclass correlation coefficients (ICCs) of 0.66 for children and 0.88 for parents (P < 0.01). Correlation coefficients between iteims and their hypothesized subscales were higher than those with other subscales (P < 0.05). The subscale of "About My/My Child's Neuromuscular Disease" significantly related to mobility and stair climbing status (Child t = 2.21, Parent t = 2.83, P < 0.05). The inter-correlations among the Chinese version of the PedsQL(TM) 3.0 Neuromuscular Module and the PedsQL(TM) 4.0 Generic Core Scales had medium to large effect sizes (P < 0.05). The child self-report scores were in moderate agreement with the parent proxy-report scores (ICC = 0.51, P < 0.05). CONCLUSIONS: The Chinese version of the PedsQLTM 3.0 Neuromuscular Module has acceptable psychometric properties. It is a reliable measure of disease-specific HRQOL in Chinese children with DMD.

[Clinical characteristics of Mycoplasma pneumoniae-associated ischemic stroke in children, and a literature review].

OBJECTIVE: To study the clinical characteristics of Mycoplasma pneumoniae (MP)-associated ischemic stroke in children. METHODS: The case of a girl with MP-associated ischemic stroke was reported, including clinical manifestations and laboratory and imaging examinations, and related literature was reviewed. RESULTS: The girl, who was suffering from a respiratory tract infection was found to have hemiplegia and aphasia which were expressed in ischemic stroke. IgM antibody to MP in serum (1∶320) and lavage fluid was positive. Pulmonary imaging showed unilateral consolidation and pleural exudate. Cerebral neuroimaging examination showed occlusion of the bilateral middle cerebral artery, mainly on the left side. The neurological symptoms and signs were recovered after comprehensive therapy with medication (azithromycin, hormone and heparin) and rehabilitation training. CONCLUSIONS: Ischemic stroke is rare but severe manifestation of central nervous system damage in children suffering from MP infection. Cerebral imaging and etiological examinations contribute to the diagnosis. Early use of macrolide antibiotics, anticoagulant and hormone may improve the prognosis.