Application of next-generation sequencing in the molecular diagnosis of Duchenne muscular dystrophy / 中国当代儿科杂志

The purpose of this study is to analyze the family's clinical data of 22 children who were given an intended clinical diagnosis of Duchenne muscular dystrophy (DMD), and to explore the clinical value of next-generation sequencing (NGS) in the molecular diagnosis of DMD. The probands were simultaneously tested by NGS for a gene panel associated with hereditary neuromuscular disease and multiplex ligation-dependent probe amplification (MLPA) for the Dystrophin gene. The exon deletion/repetition mutations of the Dystrophin gene determined by both methods were compared and the point mutations of the Dystrophin gene were verified by Sanger sequencing. Dystrophin gene mutations were found in all the 22 probands, including 14 exon deletion/repetition mutations and 8 point mutations/minor variations. The results of MLPA detection were consistent with those of NGS. The results of Sanger sequencing showed that the point mutations and minor variations determined by NGS were correct. One missense mutation (c.6290G>T), 1 nonsense mutation (c.3487C>T) and 4 minor deletion-induced frameshift mutations (c.1208delG, c.7497_7506delGGTGGGTGAC, c.9421_9422delAA and c.8910_8913delTCTC) had not been reported in the Human Gene Mutation Database, and thus were considered as novel mutations of the Dystrophin gene. The results of this study showed that NGS can detect variations in the Dystrophin gene, including exon deletion/repetition, point mutation, minor deletion and intron mutation. Therefore, NGS is of certain clinical value in the molecular diagnosis of DMD and is worthy of recommendation.

Change in serum intestinal fatty acid binding protein and its significance in children with pneumonia and gastrointestinal injury / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the change in serum intestinal fatty acid binding protein (IFABP) in children with pneumonia and its correlation with gastrointestinal injury.</p><p><b>METHODS</b>A total of 82 children with community-acquired pneumonia who were treated from January to October, 2015 were enrolled, among whom 34 had mild pneumonia and 48 had severe pneumonia. According to pediatric critical illness score (PCIS), the children with severe pneumonia were further divided into non-critical group (25 patients) and critical group (23 patients). Thirty healthy children who underwent physical examination at outpatient service were enrolled as the control group. ELISA was used to measure serum IFABP level, and the acute gastrointestinal injury (AGI) grade was determined for children with severe pneumonia. Serum IFABP level was compared between groups, and the correlations of IFABP with AGI grade and PCIS were analyzed.</p><p><b>RESULTS</b>The severe pneumonia group showed a significantly higher serum IFABP level than the control group and the mild pneumonia group (P<0.01), and the mild pneumonia group also showed a significantly higher serum IFABP level than the control group (P<0.01). The critical group showed a significantly higher serum IFABP level than the non-critical group (P<0.01). The patients with grade I-IV AGI had significantly higher serum IFABP levels than the control group (P<0.01), and the serum IFABP level increased significantly with the increasing AGI grade (P<0.01). Serum IFABP level was positively correlated with AGI grade (P<0.01) but negatively correlated with PCIS (P<0.01).</p><p><b>CONCLUSIONS</b>Children with pneumonia experience an increased serum IFABP level which can be used as a sensitive indicator for the early diagnosis of gastrointestinal injury and the evaluation of conditions in children with pneumonia.</p>

Efficacy and safety of sodium valproate in a higher dose for treating nocturnal and early-morning seizures / 中华实用儿科临床杂志

Objective To explore the efficacy and safety of sodium valproate in a higher dose in the evening for treating nocturnal and early-morning seizures.Methods A total of 69 newly diagnosed pediatric patients with nocturnal or early-morning seizures were enrolled.They were randomly distributedinto experimental group (35 cases) and control group (34 cases) and treated with sodium valproate tablets.The initial dose was little.It was gradually increased to the effective maintenance dose.With sodium valproate given twice a day,the post meridiem(PM) dose was twice the ante meridiem(AM) dose in the experimental group,while the PM/AM dose was equal in the control group.All patients were at least been followed up for 6 months.Results In the experimental group,28 cases were seizure free (80.0％),and the total effective rate was 85.7％.In the control group,20 cases were seizure free (58.8％),and the total effective rate was 64.7％.The difference in the total effective rate between the 2 groups was significant (P ＜ 0.05).No severe adverse effect was found among all patients.The incidence of daytime sleepiness (0/35 cases) in the experimental group was lower than that in the control group (2/34 cases).Conclusions A higher dose of sodium valproate in the evening for nocturnal and early-morning seizures led to better seizure control,better nocturnal sleep,less daytime somnolence,and the side effects are slight.

Changes of soluble triggering receptor expressed on myeloid cells-1 and lactate in children with purulent meningitis / 中华实用儿科临床杂志

Objective To discuss the diagnosis significance of soluble triggering receptor expressed on myeloid cells-1 (STREM-1) and lactate in children with purulent meningitis,and to investigate the changes of STREM-1,lactate in cerebrospinal fluid(CSF) of purulent meningitis before and after treatment.Methods Dry chemical method and enzyme linked immunosorbent assay(ELISA) were used to measure STREM-1 and lactate levels in CSF of purulent meningitis group (24 cases),viral meningitis group (27 cases),CSF normal group (25 cases) and purulent meningitis after treatment group(22 cases).Results 1.STREM-1 and lactate levels in CSF were higher in patients with purulent meningitis than in those with viral meningitis and CSF normal group(all P ＜ 0.05).2.STREM-1 and lactate levels in CSF were higher in patients with purulent meningitis before treatment than those after treatment(all P ＜ 0.05).3.The area under the curve(AUC) of STREM-1 in CSF was 0.891,and at a cutoff level of 27.86 ng/L STREM-1 yielded a sensitivity of 80.8％ and specificity of 75.0％ ;the AUC of CSF lactate was 0.940,and at a cutoff level of 1.75 mmol/ L lactate yielded a sensitivity of 90.4％ and specificity of 83.3％.Conclusions 1.STREM-1 and lactate were associated with bacterial infection,they have considerable diagnostic values in purulent meningitis.2.STREM-1 and lactate maybe worthless in differential diagnosis of purulent meningitis when treated by effective antibiotics.3.The decline of STREM-1 and lactate in CSF prompts the control of infection and good prognosis.

Hydroa vacciniforme-like cutaneous T cell lymphoma: a case report and literature review / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical features, diagnosis and therapy of hydroa vacciniforme-like cutaneous T cell lymphoma.</p><p><b>METHODS</b>The clinical presentations and the findings of laboratory examinations and skin biopsy of affected tissue in a child with hydroa vacciniforme-like cutaneous T cell lymphoma were retrospectively reviewed.</p><p><b>RESULTS</b>The child manifested as rash, fever and lymph node intumesce. Rash was pantomorphia, including edematous erythema, vesicles, crusts, necrosis and depressed scar, and it was mild in winter and severe in summer, mainly involving in the face and extremities. Epstein-Barre virus (EBV)-IgM was positive. Histopathological findings revealed focal lymphocyte invasion in subcutaneous panniculus adiposus, mainly surrounding the blood vessels. Immunohistochemistry showed CD3 (+), CD43 (+), CD20 (-), pax-5 (-), TIA (+), CD5 (+), CD8 (+), Granmye (+) and CD4 (-). The clinical symptoms were improved after glucocorticoid treatment in this child.</p><p><b>CONCLUSIONS</b>Hydroa vacciniforme-like cutaneous T cell lymphoma has special clinical manifestations. This disorder may be definitely diagnosed by skin biopsy of affected tissue and immunohistochemistry assay. Glucocorticoid treatment is effective. EBV infection may be related to the development of this disorder.</p>

Ten-year changes in pathogen, antimicrobial susceptibility and clinical feature of children with bacterial meningitis / 中华儿科杂志

<p><b>OBJECTIVE</b>Despite progress in antibiotic therapy and intensive care, childhood bacterial meningitis (BM) remains a devastating disease. We conducted this study to investigate the changes in clinical characteristics, the etiologic agents and antimicrobial susceptibility of BM during the past 10 years in children under 14 years of age.</p><p><b>METHODS</b>These 126 patients were divided into two groups according to their date of admission. Group 1 included 64 patients admitted from January 1998 to December 2002, and group 2 included 62 cases admitted from January 2003 to December 2007. All pediatric medical charts of them were reviewed.</p><p><b>RESULTS</b>The predominant isolated bacteria from CSF were coagulase-negative staphylococcus (17/62, 27.4%) and Escherichia coli (9/62, 14.5%) in group 2. The resistance rate of staphylococcus against oxacillin (MRS) was 68.4% (13/19) in group 2, significantly higher than that of group 1 (16.7%, 2/12). Among 126 cases, 42 had seizure attack and 16 had consciousness disturbance, the proportions of them in group 2 (11/62, 17.7%; 4/62, 6.4%) were lower than those in group 1 (31/64, 48.4%; 12/64, 18.8%, P < 0.05). Cases in group 2 survived with complications [13/62 (21.0%)] and sequelae [11/62 (17.7%)] were lower than those in group 1 (24/64, 37.5%, 23/64, 35.9%, P < 0.05), but the rate of empirical therapy modification in group 2 (21/62, 33.9%) was higher than that in group 1 (7/64, 10.9%, P < 0.01).</p><p><b>CONCLUSION</b>The predominant bacteria in children with BM are staphylococcus and Escherichia coli in recent years. The antibiotic resistance rate of bacteria has been higher year after year. The clinical patterns of pediatric BM have changed with a decrease in clinically serious cases, complications and sequelae, but an increase in modification of empirical therapy.</p>

Effect of Ethyl Pyruvate on the Level of High Mobility Group Box 1 in Brain Injury of Infant Rats Induced by Lipopolysaccharide and Its Significance / 实用儿科临床杂志

Objective To investigate the influence of Ethyl pyruvate(EP) on the level of high mobility group box 1 (HMGB1) in brain injury of infant rats induced by lipopolysaccharide (LPS) and its significance.Methods Two hundred and forty normal healthy 1-month-old Spragne-Dawley (SD) rats were randomly divided into 3 groups:9 g/L sodium chloride (NS) group(n=80),LPS group (n=80),and EP group (n=80).LPS (1 mg/kg) was injected via internal carotid.In EP group,after injecting LPS,each rat was immediately administrated 4 mL EP(40 mg/kg) intraperitoneally; in control group,4 mL Ringer's solution was given instead of EP.Rats were decapitated at 6,12,24,48 and 72 h following drug injection.The evan's blue (EB) content of brain tissues was meteraged by the formamide methods.Immunohistochemistry technology was used to detect the expression of HMGB1,neuron specific enolase(NSE) and glial fibrillary acidic protein(GFAP) protein,and reverse transcribe polymerase chain reaction technology was applied to study the expression level of HMGB1 mRNA in brain tissue.Meanwhile,the pathological changes of brain tissues were observed under the light microscope.Results Six hours after LPS was given,brain EB content,the levels of NSE,GFAP protein started to rise,reaching the peak at 24 h,and still higher than those in control group at 48 h and 72 h.The expression pattern of HMGB1 protein and mRNA in brain tissue was consistent with the severity of brain injury,increased at 6 h after LPS was given and reached the peak at 24 h,still higher than those in control group at 48 h and 72 h.Positive correlation was found among HMGB1 protein,HMGB1 mRNA and EB content,NSE protein,GFAP protein,respectively.In EP group,the levels of HMGB1 protein and mRNA,the levels of brain EB content,NSE,GFAP protein were found,positive correlation was still gotten between HMGB1 protein,EB content and NSE,GFAP protein,HMGB1 mRNA in LPS group and EP group.Conclusion EP has neuroprotective effect on brain injury induced by LPS,which may be relevant to decreasing of the expression of HMGB1 and suppressing inflammation action.

Inhibitory Effect of Melatonin on Expression of Glial Fibrillary Acidic Protein in Infant Rats with Brain Injury Induced by Lipopolysaccharide / 实用儿科临床杂志

Objective To explore the inhibitory effect of melatonin(MT) on expression of glial fibrillary acidic protein in infant rats with brain injury induced by lipopolysaccharide(LPS).Methods One hundred and sixty Spragne-Dawley rats(1 month) were randomly divided into 4 groups:9 g/L saline group(NS group),LPS group,MT protective A group(MT plus LPS,MT was given 15 minutes earlier),and MT protective B group(MT plus LPS,MT was given immediately).Forty rats in each group,then every group was subdivided into 2 subgroups according to observe time point at 24 h and 48 h with 20 rats,respectively.Half of them in all subgroups were not injected Evans blue(EB).Rats were sacrificed at 2 time points to take brain tissue samples,respectively.EB content of brain tissue was meteraged by the formamide methods.Immunohistochemistry technology and computer assisted image analysis system methods were applied to observe the expression of the positive apoptosis cell of brain tissue.Results Both EB content and the expression of GFAP showed significant difference between LPS group and NS group(Pa0.05).Conclusions LPS can induce the expression of GFAP in infectious brain injury of rats induced by LPS,and MT can inhibit the expression of GFAP;MT may play a role in dealing with infectious brain injury.

Analysis of 262 Cases of Seizures Detected with Video-Electroencephalogram / 实用儿科临床杂志

Objective To evaluate the clinical significance of video-electroencephalogram(VEEG) in the diagnosis of paroxysmal events in children.Methods The VEEG and routine electroencephalogram(EEG) were used to inspect 262 cases of seizures respectively collected of outpatient clinic and admission department from Apr.2006 to Dec.2007,and VEEG monitoring results were analyzed and compared with primary diagnoses retrospectively.According to the characteristics of EEG and clinical observation to decide whether it be epilepsy or not and compare the difference between the results of EEG and VEEG to evaluate the application value of the 2 examination methods in children with NES.SPSS 13.0 software was used to analyze the data.Results Of all 262 cases,113 cases of clinical outbreak were recorded,meanwhile the diagnose of 4 patients of epileptic seizures(ES) group were changed to non-epileptic seizures(NES) and 3 patients of NES were rediagnosed to ES,69 cases couldn′t be confirmed according to VEEG or EEG and there was no outbreak recorded in the other 149 cases.In the period onset,79 children′s characteristics of EEG were captured which included 76 children in ES group,and 3 children in NES group.Thirty-six children′s characteristics of EEG were not captured which included 4 children in ES group and 30 children in NES group.The differences of the ratio of total detect and abnormity between EEG and VEEG were significant,improving the diagniosis rate and the control rate of symptom.VEEG had a clinical significance in differential diagnosis of ES and NES.Conclusions Compared with EEG,VEEG will be more helpful in diagnosing and differentiating seizures diseases,improving the diagnosis rate and the control rate of symptom.VEEG has a clinical in diffe-rential diagnosis for ES and NES.

Case Report of Mixed Connective Tissue Disease Complicating Pulmonary Hypertension and Its Literature Review / 实用儿科临床杂志

Objective To investigate the diagnosis and treatment of mixed connective tissue disease(MCTD)complicating pulmonary hypertension(PAH) in childhood in order to improve the recognition of this disease.Method According to the symptoms,signs,past history,labratory examinations,the child′s disease was diagnosed and treated,and the relative literature was reviewed.Results The main symptom of the child was interruptable apsychia.Ultrasound showed severe PAH,positive of anti-RNP antibody.After given immunosuppressant and decreased PAH,the patient′s condition was more improved.Conclusions MCTD complicating PAH in childhood onstes delitescently,and it′s difficult to diagnose.Recognition should be elevated to diagnose and treat it earlier.The prognosis can be improved.

Expression of Intercellular Adhesion Molecule-1 in Brain Edema Induced by Lipoposacchride in Rats under Action of Dexamethasone / 实用儿科临床杂志

Objective To explore the expression of intercellular adhesion molecule-1(ICAM-1) in the brain edema induced by lipoposacchride(LPS) in rats under the action of dexamethasone.Methods One hundred and fifty healthy SD rats were randomly divided into three groups of 50 each:normal saline group(NS group),LPS group and dexamethasone group(DXM group).Each group were again divided into 5 groups:4,6,12,24 and 48 h group.Brain edema was induced by LPS.Immunohistochemistry staining methods were used to measure the expression of ICAM-1 in brain tissue of brain edema induced by LPS in rats.And the level of evans blue(EB) was aslo determined.Results At each time point,the content of brain tissue and evans blue(EB) in LPS and DXM group all increased significantly than those in NS group(Pa0.05).In LPS group,brain water content and EB content,expressing quantity of ICAM-1 and brain water content,expressing quantity of ICAM-1 and EB content all had positive relationship(r=0.537,0.467,0.549 Pa

Clinical study of 83 cases with spinal muscular atrophy in children / 中华儿科杂志

<p><b>OBJECTIVE</b>Spinal muscular atrophy (SMA) is a common autosomal recessive disorder and represents one of the most common genetic causes of death in childhood. The last 10 years have seen major advances in the field of SMA, but no curative treatment is available so far. This study aimed to analyze the clinical characteristics of SMA, improve the clinical diagnosis of SMA, and explore the importance of gene diagnosis and prenatal diagnosis of SMA by gene deletion analysis.</p><p><b>METHODS</b>Totally 83 cases with SMA including 55 males and 28 females were enrolled in this study. The age was between 1 day and 14 years (average 23.7 months). The clinical characteristics and changes of electromyography were assessed in all cases. The muscular biopsy was performed in 2 of 83 cases. The deletion of survival of motor neuron gene (SMN) was detected by PCR and restriction endonuclease spectrum analysis in 13 of 83 cases.</p><p><b>RESULTS</b>The 83 cases were subdivided into three clinical groups based on age of onset of symptom, age at death and achievement of certain motor milestone, 60 cases with type I, 19 cases with type II and 4 cases with type III. They were all characterized by symmetric muscle weakness (more proximal than distal) associated with atrophy, absence or marked decrease of deep tendon reflexes. Electromyographic studies showed a pattern of denervation with neither sensory involvement nor marked decrease of motor nerve conduction velocities in all cases. Muscle biopsy provided evidence of skeletal muscle denervation with groups of atrophy in 2 cases. The SMN detection revealed deletion of exon 7 and exon 8 in 11 of 13 cases, only lacking exon 7 in 1 of 13 cases and lacking exon 8 in 1 of 13 cases.</p><p><b>CONCLUSION</b>SMA is characterized by degeneration of lower motor neuron associated with muscle paralysis and atrophy. The definite diagnosis of SMA will rely on the typical clinical characteristics, changes of electromyogram and muscle biopsy and gene deletion analysis. Gene diagnosis of SMA can provide a basis for prenatal diagnosis which is of great importance in preventing SMA.</p>

Relationship between Spondyloppiphyseal Dysplasia Tarda Gene Escaping X Chromosome Inactivation and Spondyloppiphyseal Dysplasia Tarda Phenotype / 实用儿科临床杂志

Objective To explore the relationship between X - linked spondyloepiphyseal dysplasia tarda (SEDL) gene escaping X chromosome inactivation( XCI) and SEDL phenotype. Methods RT - PCR was performed on total RNA which was isolated from blood samples of patients, female carriers and controls. Patients and female carriers were selected from the pedigree with SEDL caused by the mutation (IVS2 - 2A→C) of the gene. cDNA was analyzed by polyacrylamide gelelectrophoresis(PAGE). Results PAGE data indicateed that female carriers expressed both normal and mutant SEDL mRNA,meaning the SEDL gene escaping XCI. Family investigation showed carrier females in the SEDL pedigree presented no symptoms. Conclusions The SEDL gene escaping X chromosome in-activation is firstly identified from human body. This may explain that carrier females present no symptoms.

Gene diagnosis of X-linked spondyloepiphyseal dysplasia tarda by linkage analysis and DNA sequencing / 中华儿科杂志

<p><b>OBJECTIVE</b>X linked spondyloepiphyseal dysplasia tarda (SEDL) is heritable osteochondrondysplasia characterized in affected males by disproportional short stature with short neck and trunk resulting from a growth defect of the vertebral bodies, accompanied by barrel chest and degenerative osteoarthropathy of hip joints. This progressive skeletal dysplasia is caused by the SEDL gene located approximately 100 kb centromeric of DXS16 at Xp22. The disorder usually manifests in late childhood without systemic complications, and generally female carriers of SEDL are asymptomatic. So the diagnosis of potential carriers and presymptomatic patients is almost impossible. This study aimed to establish methods of gene diagnosis for finding out potential carriers and presymptomatic patients.</p><p><b>METHODS</b>The blood samples were collected from 21 individuals in a large Chinese pedigree with SEDL. Microsatellite marker DXS16 was selected for linkage analysis. In order to confirm the allele of DXS16 linked to the pathogenic SEDL gene, polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) were used to examine the variability of the lengths of DXS16, and linkage analysis was performed for the diagnosis of potential carriers and presymptomatic patients. Then the pathogenic mutation of the SEDL gene in the family was identified by bi-directionally direct sequencing of PCR products amplified for each of the four coding exons as well as their exon/intron boundaries. The potential carriers and presymptomatic patients were also diagnosed in this way.</p><p><b>RESULTS</b>Six young individuals (IV(14), IV(19), IV(21), IV(23), V(4), V(7))who wanted to know whether they were carriers or presymptomatic patients were diagnosed by linkage analysis. Four females of them (IV(14), IV(19), IV(21), V(7)) were determined being carriers because they carry the allele of DXS16 which links the pathogenic SEDL gene, and the other two (IV(23), V(4)) being normal individuals for their alleles of DXS16 linked with wild SEDL gene. DNA sequencing identified that the pathogenic mutation of SEDL gene in the family, which was a nucleotide substitution of the splice-acceptor site in intron 2, IVS2 -2 A-->C. This is a novel mutation in the SEDL gene. Four female individuals (IV(14), IV(19), IV(21), V(7)) carried the mutation; individuals IV(23) and V(4) carried the wild SEDL gene. The results of diagnosis of linkage analysis coincide completely with that of DNA sequencing.</p><p><b>CONCLUSION</b>Linkage analysis is a simple, rapid and inexpensive gene diagnosis method for SEDL and its accuracy was the same as DNA sequencing. Each of linkage analysis and DNA sequencing can be used to diagnose SEDL, which is very helpful for finding potential carriers and presymptomatic patients.</p>

Identification of a novel mutation IVS2-2A-->C of SEDL gene in a Chinese family with X-linked spondyloepiphyseal dysplasia tarda / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutation of spondyloepiphyseal dysplasia tarda (SEDL) gene in a large Chinese family with X-linked spondyloepiphyseal dysplasia tarda and to make a discussion on the pathogenesis of SEDL at the molecular level.</p><p><b>METHODS</b>In two patients, four exons comprising the SEDL open reading frame as well as their exon/intron boundaries were analyzed by bi-directional direct sequencing of PCR products. The sequencing results were compared against the normal sequences in GenBank to find the mutation. Then the mutation was identified in other members of the family.</p><p><b>RESULTS</b>A nucleotide substitution of the splice acceptor in SEDL intron 2, IVS2 -2A-->C,was detected in two affected individuals (IV(15) V(3)) in the Chinese family with SEDL, but no sequence change occurring on exons 3-6 was detected. The transversion was also identified in four heterozygous carriers. The mutation was not found in two unaffected male individuals and fifteen normal controls. Furthermore, four potential carriers were identified in the family.</p><p><b>CONCLUSION</b>The mutation IVS2 -2A-->C of SEDL gene was firstly determined in the world. The change of the splice acceptor in SEDL intron 2 may cause skipping of exon 3 which is responsible for the disease. Molecular diagnosis can be made by detecting the mutation.</p>

Expression of Macrophage Inflammatory Protein 2 in Brain Edema Caused by Lioposacchride in Rats / 实用儿科临床杂志

Objective To study the expression of macrophage inflammatory protein 2(MIP-2) and the interfering effects of naloxone in the brain edema caused by lioposacchride (LPS)in rats.Methods Eithty-four SD rats were randomly divided into 3 groups:normal saline group(NS group,n=28) 0.2 mL normal saline was injected by carotid into each rat;LPS group(n=28) with 200 ?g LPS;naloxone interfering group(NAL group,n=28)1 mg/kg naloxone was intraperitoneally injected at 10 min,1,2,6,12 h and following LPS injected 2 h before decapitation.The content of MIP-2 and even blue(EB) in brain tissue were detected at different time point.The brain water content was measured by drying method.Results The content of water and EB in LPS group were significan higher than those in NS group(P