Metagenomic next-generation sequencing and proteomics analysis in pediatric viral encephalitis and meningitis.

Introduction: Early and accurate identification of pathogens is essential for improved outcomes in patients with viral encephalitis (VE) and/or viral meningitis (VM). Methods: In our research, Metagenomic next-generation sequencing (mNGS) which can identify viral pathogens unbiasedly was performed on RNA and DNA to identify potential pathogens in cerebrospinal fluid (CSF) samples from 50 pediatric patients with suspected VEs and/or VMs. Then we performed proteomics analysis on the 14 HEV-positive CSF samples and another 12 CSF samples from health controls (HCs). A supervised partial least squaresdiscriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) model was performed using proteomics data. Results: Ten viruses in 48% patients were identified and the most common pathogen was human enterovirus (HEV) Echo18. 11 proteins overlapping between the top 20 DEPs in terms of P value and FC and the top 20 proteins in PLS-DA VIP lists were acquired. Discussion: Our result showed mNGS has certain advantages on pathogens identification in VE and VM and our research established a foundation to identify diagnosis biomarker candidates of HEV-positive meningitis based on MS-based proteomics analysis, which could also contribute toward investigating the HEV-specific host response patterns.

Proteomic profiling of cerebrospinal fluid in pediatric myelin oligodendrocyte glycoprotein antibody-associated disease.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an autoimmune demyelinating disorder of the central nervous system. METHODS: Extracted proteins from 34 cerebrospinal fluid (CSF) samples [patients with MOGAD (MOG group, n = 12); healthy controls (HC group, n = 12); patients with MOG seronegative and metagenomics next-generation sequencing-negative inflammatory neurological diseases (IND group, n = 10)] were processed and subjected to label-free quantitative proteomics. Supervised partial least squares-discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) models were also performed based on proteomics data. Functional analysis of differentially expressed proteins (DEPs) was performed using Gene Ontology, InterPro, and Kyoto Encyclopedia Genes and Genomes. An enzyme-linked immunosorbent assay was used to determine the complement levels in serum from patients with MOGAD. RESULTS: Four hundred and twenty-nine DEPs (149 upregulated and 280 downregulated proteins) were identified in the MOG group compared to the HC group according to the P value and fold change (FC). Using the O-PLS-DA model, 872 differentially abundant proteins were identified with variable importance projection (VIP) scores > 1. Five proteins (gamma-glutamyl hydrolase, cathepsin F, interalpha-trypsin inhibitor heavy chain 5, latent transforming growth factor beta-binding protein 4 and leukocyte-associated immunoglobulin-like receptor 1) overlapping between the top 30 DEPs with top-ranked P value and FC and top 30 proteins in PLS-DA VIP lists were acquired. Functional analysis revealed that the dysregulated proteins in the MOG group were primarily involved in complement and coagulation cascades, cell adhesion, axon guidance, and glycosphingolipid biosynthesis compared to the HC group. CONCLUSION: The proteomic alterations in CSF samples from children with MOGAD identified in the current study might provide opportunities for developing novel biomarker candidates.

A child with household transmitted COVID-19.

BACKGROUND: Although people of all ages are susceptible to the novel coronavirus infection, which is presently named "Coronavirus Disease 2019" (COVID-19), there has been relatively few cases reported among children. Therefore, it is necessary to understand the clinical characteristics of COVID-19 in children and the differences from adults. CASE PRESENTATION: We report one pediatric case of COVID-19. A 14-month-old boy was admitted to the hospital with a symptom of fever, and was diagnosed with a mild form of COVID-19. The child's mother and grandmother also tested positive for SARS-CoV-2 RNA. However, the lymphocyte counts were normal. The chest computed tomography (CT) revealed scattered ground glass opacities in the right lower lobe close to the pleura and resorption after the treatment. The patient continued to test positive for SARS-CoV-2 RNA in the nasopharyngeal swabs and stool at 17 days after the disappearance of symptoms. CONCLUSION: The present pediatric case of COVID-19 was acquired through household transmission, and the symptoms were mild. Lymphocyte counts did not significantly decrease. The RNA of SARS-CoV-2 in stool and nasopharyngeal swabs remained positive for an extended period of time after the disappearance of symptoms. This suggests that attention should be given to the potential contagiousness of pediatric COVID-19 cases after clinical recovery.

Insulin-Like Growth Factor-1 Down-Regulates the Phosphorylation of FXYD1 and Rescues Behavioral Deficits in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is a neurodevelopmental disease in children that is mainly caused by mutations in the MeCP2 gene, which codes for a transcriptional regulator. The expression of insulin-like growth factor-1 (IGF-1) is reduced in RTT patients and animal models, and IGF-1 treatment is a promising therapeutic strategy for RTT. However, the mechanism underlying the effects of IGF-1 remains to be further explored. FXYD1 is an auxiliary subunit of Na, K-ATPase. Overexpression of FXYD1 is involved in the pathogenesis of RTT. However, whether IGF-1 exerts its effect through normalizing FXYD1 is completely unknown. To this end, we evaluated the effect of IGF-1 on FXYD1 expression and posttranslational modification in a mouse model of RTT (MeCP2308) using both in vitro and in vivo experiments. The results show that FXYD1 mRNA and phosphorylated protein (p-FXYD1) were significantly elevated in the frontal cortex in RTT mice, compared to wild type. In RTT mice, IGF-1 treatment significantly reduced levels of FXYD1 mRNA and p-FXYD1, in parallel with improvements in behavior, motor coordination, and cognitive function. For mechanistic insight into the effect of IGF-1 on p-FXYD1, we found the decreased phosphorylated forms of PI3K-AKT-mTOR signaling pathway components in the frontal cortex of RTT mice and the normalizing effect of IGF-1 on the phosphorylated forms of these components. Interestingly, blocking the PI3K/AKT pathway by PI3K inhibitor could abolish the effect of IGF-1 on p-FXYD1 level, in addition to the effect of IGF-1 on the phosphorylation of other components in the PI3K/AKT pathway. Thus, our study has provided new insights into the mechanism of IGF-1 treatment for RTT, which appears to involve FXYD1.

[A genotyping study of 13 cases of early-onset Charcot-Marie-Tooth disease].

OBJECTIVE: To study the clinical characteristics and genetic variation of early-onset Charcot-Marie-Tooth disease (CMT). METHODS: Children with a clinical diagnosis of early-onset CMT were selected for the study. Relevant clinical data were collected, and electromyogram and CMT-related gene detection were performed and analyzed. RESULTS: A total of 13 cases of early-onset CMT were enrolled, including 9 males (69%) and 4 females (31%). The mean age at consultation was 4.0±2.1 years. Among them, 12 children (92%) had an age of onset less than 2 years, 9 children (69%) were diagnosed with CMT type 1 (including 6 cases of Dejerine-Sottas syndrome), 1 child (8%) with intermediate form of CMT, and 3 children (23%) with CMT type 2. The genetic test results of these 13 children showed 6 cases (46%) of PMP22 duplication mutation, 3 cases (23%) of MPZ gene insertion mutation and point mutation, 3 cases (23%) of MFN2 gene point mutation, and 1 case (8%) of NEFL gene point mutation. Eleven cases (85%) carried known pathogenic mutations and 2 cases (15%) had novel mutations. The new variant c.394C>G (p.P132A) of the MPZ gene was rated as "possibly pathogenic" and the new variant c.326A>G (p.K109R) of the MFN2 gene was rated as "pathogenic". CONCLUSIONS: Early-onset CMT is mainly caused by PMP22 gene duplication mutation and MPZ gene mutations. The clinical phenotype is mainly CMT type 1, among which Dejerine-Sottas syndrome accounts for a considerable proportion.

Clinically mild encephalitis/encephalopathy with a reversible splenial lesion associated with Mycoplasma pneumoniae infection.

BACKGROUND: Clinically mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) is a clinico-radiological syndrome characterized by transient mild symptoms of encephalopathy and a reversible lesion in the splenium of the corpus callosum on magnetic resonance imaging (MRI). It is often triggered by infection. The common pathogens of MERS are viruses, especially influenza virus. However, Mycoplasma pneumoniae (M.pneumoniae) are relatively rare pathogens for MERS. CASE PRESENTATION: Here we report two paediatric cases of M.pneumoniae infection-induced MERS. The diagnosis of M.pneumoniae infection was established based on polymerase chain reaction (PCR) and specific serum antibodies (IgM). Both of the two patients presented with mild encephalopathy manifestations and recovered completely within a few days. The initial MRI showed a lesion in the central portion of the splenium of the corpus callosum, which completely resolved on the seventh and eighth day after admission for case 1 and case 2. Lumbar puncture was performed in both patients, which revealed no pleocytosis. In case 1, the patient had hyponatremia, peripheral facial nerve paralysis, and rash. To the best of our knowledge, it is the first MERS case associated with peripheral nerve damage. In case 2, interleukin-6(IL-6) was moderately increased in the cerebrospinal fluid (CSF). It suggested that IL-6 may play a role in the pathogenesis of M.pneumoniae-induced MERS. CONCLUSION: Our study enriches the available information on the pathogens of MERS and provides valuable data for better understanding of this syndrome.

Reversible bilateral striatal lesions following Mycoplasma pneumoniae infection associated with elevated levels of interleukins 6 and 8.

BACKGROUND: Reversible bilateral striatal necrosis associated with Mycoplasma pneumoniae (M. pneumoniae) infection is a rare neurological disease. The exact pathogenic mechanism remains unknown. PATIENT: We report reversible bilateral striatal lesions with a favorable outcome secondary to M. pneumoniae infection in an 8-year-old Chinese girl. Cranial MRI showed abnormal signals in bilateral striatum, which disappeared 8 months later. To better understand the pathogenesis of this encephalopathy, we examined cytokines levels in serum and cerebrospinal fluid from this patient. The results revealed the concentrations of interleukin-6 and interleukin-8 increased significantly in serum (26 pg/mL and 66 pg/mL, respectively) and cerebrospinal fluid (122 pg/mL and 325 pg/mL, respectively), and were reduced markedly after the therapy. Intrathecal production of interleukin-6 and interleukin-8 is probably related to the pathogenesis of striatal lesions caused by M. pneumoniae. These cytokines may cause local vascular injury, and finally leading to local vascular occlusion. CONCLUSION: Our results suggest that interleukin-6 and interleukin-8 may play important roles in the pathogenesis of this disease. This is the first report to describe the role of cytokines in this condition and relevant literature is reviewed. Our findings may lead to better understanding of the pathogenesis of M. pneumoniae-associated striatal lesions.

Novel mutations of the ATP7B gene in Han Chinese families with pre-symptomatic Wilson's disease.

BACKGROUND: Wilson's disease (WD) is an autosomal recessive genetic disorder of copper metabolism, caused by mutations in the ATP7B gene, resulting in copper accumulation in the liver, brain, kidney, and cornea and leading to significant disability or death if untreated. Early diagnosis and proper therapy usually predict a good prognosis, especially in pre-symptomatic WD. Genetic testing is the most accurate and effective diagnostic method for early diagnosis. METHODS: The clinical and biochemical features of three unrelated Han Chinese families with pre-symptomatic WD were reported. The molecular defects in these families were investigated by polymerase chain reaction and DNA sequencing. Hundred healthy children with the same ethnic background served as controls. Bioinformatic tools (polymorphism phenotyping-2, sorting intolerant from tolerant, protein analysis through evolutionary relationships, and predictor of human deleterious single nucleotide polymorphisms) were combined and used to predict the functional effects of mutations. RESULTS: We identified 2 novel ATP7B mutations (p.Leu692Pro and p.Asn728Ser) and 3 known mutations (p.Met769fs, p.Arg778Leu and p.Val1216Met) in these Chinese WD families. These mutations were not observed in the 100 normal controls. The bioinformatic method showed that p.Leu692Pro and p.Asn728Ser mutations are pathogenic. CONCLUSIONS: Our research enriches the mutation spectrum of the ATP7B gene worldwide and provides valuable information for studying the mutation types and mode of inheritance of ATP7B in the Chinese population. Liver function analysis and genetic testing in young children with WD are necessary to shorten the time to the initiation of therapy, reduce damage to the liver and brain, and improve prognosis.

Novel chloride channel gene mutations in two unrelated Chinese families with myotonia congenita.

Myotonia congenita (MC) is a genetic disease characterized by mutations in the muscle chloride channel gene (CLCN1). To date, approximately 130 different mutations on the CLCN1 gene have been identified. However, most of the studies have focused on Caucasians, and reports on CLCN1 mutations in Chinese population are rare. This study investigated the mutation of CLCN1 in two Chinese families with MC. Direct sequencing of the CLCN1 gene revealed a heterozygous mutation (892G>A, resulting in A298T) in one family and a compound heterozygous mutations (782A>G, resulting in Y261C; 1679T>C, resulting in M560T) in the other family, None of the 100 normal controls had these mutations. Our findings add more to the available information on the CLCN1 mutation spectrum, and provide a valuable reference for studying the mutation types and inheritance pattern of CLCN1 in the Chinese population.

[Efficacy of chemotherapy using 10-hydroxycamptothecin on recurrent or refractory neuroblastoma in children].

OBJECTIVE: The patients with recurrent or refractory neuroblastoma have a very poor prognosis and high mortality. 10-hydroxycamptothecin (HCPT) is a new agent extracted from comptotheca acuminate, a native plant. It has been shown to be very effective in some solid tumors such as gastric and colon cancers, lung cancers and ovary cancers. However, its efficacy in neuroblastoma has not been determined. This study aimed to investigate the therapeutic effects of HCPT in the treatment of recurrent or refractory neuroblastoma in children. METHODS: Ten children with recurrent neuroblastoma and two children with refractory neuroblastoma were treated with HCPT. Of them, 5 children with recurrent neuroblastoma were treated with HCPT alone, and the other 7 patients received combination chemotherapy of HCPT plus other agents. The HCPT alone treatment group was injected with HCPT (7.5 mg/m2 daily) for 14 consecutive days. The combination chemotherapy group was alternately treated with the modified new protocol A1 (cyclophosomide 1 200 mg/m2 on day 1, etoposide 100 mg/m2 on days 1-5, HCPT 5 mg/m2 on days 1-3, cisplatin 90 mg/m2 on day 4) and the modified protocol B (ifosfomide 1.5 g/m2 on days 1-5, HCPT 5 mg/m2 on days 1-3, carboplatin 450 mg/m2 on day 2). RESULTS: Four patients (33.3%) achieved partial remission and 8 patients (66.7%) had stable disease. The median remission duration was 3.5 months (2-5 months). HCPT treatment as a single agent resulted in mild side effects. Myelosuppression and digestive disorders were found as the main adverse events in the combined chemotherapy group. No chemotherapy related deaths were found. CONCLUSIONS: HCPT is safe and effective in the treatment of recurrent or refractory neuroblastoma. The toxicities of HCPT are tolerable. The long-term efficacy of HCPT warrants further research.

Thyrotropin receptor and thyroid transcription factor-1 genes variant in Chinese children with congenital hypothyroidism.

The aim of the present study was to investigate the mutation/variant of thyrotropin receptor (TSHR) and thyroid transcription factor-1 (TTF-1) genes in Chinese children with congenital hypothyroidism (CH). Seventy-nine and forty-nine Chinese children with CH were enrolled for molecular analysis of the TSHR gene and TTF-1 gene, respectively. One hundred normal children were evaluated as control. The coding regions of TSHR and TTF-1 genes were amplified by polymerase chain reaction and sequenced. Sequencing of the TSHR gene revealed a compound heterozygous variants (Pro52Thr/Val689Gly) and a heterozygous variant (Gly245Ser) in 2 of 79 patients. In 30 patients and 33 controls the normal cytosine at position 2181 in exon 10 of TSHR gene was replaced by a guanineCresulting in the replacement of Asp (727) by Glu. In 47 patients and 50 controls, the normal thymidine at position 561 in exon 7 of TSHR gene was replaced by a cytosine. This substitution did not change the amino acid in position 187. Sequencing of the TTF-1 gene revealed no mutation or polymorphism in 49 patients and 100 controls. In conclusion, three heterozygous variants (Pro52Thr, Gly245Ser, Val689Gly) of TSHR gene were firstly detected in Chinese children with CH. There were polymorphisms in exon 10 at nucleotide 2181 (C/G) and in exon 7 at nucleotide 561 (T/C) in TSHR gene. No mutation or polymorphism was detected in the coding region of TTF-1 gene. The mutation/variant of TSHR and TTF-1 genes is relatively rare in Chinese children with CH.

[Thyrotropin receptor gene inactivating mutation in Chinese children with congenital hypothyroidism].

OBJECTIVE: The inactivating mutation of thyrotropin receptor (TSHR) gene results in partial or complete insensitivity of thyrotropin (TSH) and dysfunction of the TSH-TSHR-cAMP cascade. Therefore, it may cause congenital hypothyroidism (CH). Depending on the degree of impairment of TSHR function, patients can present with subclinical hypothyroidism at one extreme of the spectrum, or severe hypothyroidism at the other. This study aimed to understand the relation between inactivating mutations of TSHR gene and Chinese children with CH. METHODS: (1) Seventy-nine Chinese children with CH, including 14 subclinical hypothyroidism patients (8 boys and 6 girls, age 1 - 5.5 years) and 65 hypothyroidism patients (27 boys and 38 girls, age 1.5 - 6 years) were enrolled in this study. Meanwhile, 100 normal children were enrolled as control, 40 were male and 60 were female. The age of the normal children were at a range of 1 - 8 years. (2) Total genomic DNA was extracted from peripheral blood leukocytes of the 79 patients and 100 normal subjects. Exons 1 - 10 of TSHR gene were individually amplified by polymerase chain reaction (PCR) and mutations were detected by direct sequencing. RESULTS: (1) A compound heterozygous missense mutations (Pro52Thr/Val689Gly) and a heterozygous missense mutation (Gly245Ser) were detected in 79 patients. The mutations of Pro52Thr and Gly245Ser were located within the extracellular domain of TSHR, while Val689Gly was located within the intracellular domain of TSHR. In 30 patients the normal cytosine at position 2181 in exon 10 was replaced by a guanine (GAC-->GAG), resulting in the replacement of Glu(727) by Asp. In 47 patients, the normal thymidine at position 561 in exon 7 was replaced by a cytosine (AAT-->AAC). This substitution did not change the amino acid (Asn) at position 187. (2) In 33 normal children the normal cytosine at position 2181 in exon 10 was also replaced by a guanine (GAC-->GAG) and in 50 normal children the normal thymidine at position 561 in exon 7 was replaced by a cytosine (AAT-->AAC). CONCLUSIONS: Three heterozygous missense mutations (Pro52Thr, Gly245Ser, Val689Gly) of TSHR gene were firstly detected in Chinese children with CH. There was a polymorphism in exon 10 at nucleotide 2181 (GAC-->GAG) and in exon 7 at nucleotide 561 (AAT-->AAC) in TSHR gene. The inactivating mutation of TSHR gene is an infrequent pathogeny for CH.