Novel collagen VI mutations identified in Chinese patients with Ullrich congenital muscular dystrophy.

BACKGROUND: We determined the clinical and molecular genetic characteristics of 8 Chinese patients with Ullrich congenital muscular dystrophy (UCMD). METHODS: Clinical data of probands were collected and muscle biopsies of patients were analyzed. Exons of COL6A1, COL6A2 and COL6A3 were analyzed by direct sequencing. Mutations in COL6A1, COL6A2 and COL6A3 were identified in 8 patients. RESULTS: Among these mutations, 5 were novel [three in the triple helical domain (THD) and 2 in the second C-terminal (C2) domain]. We also identified five known missense or in-frame deletion mutations in THD and C domains. Immunohistochemical studies on muscle biopsies from patients showed reduced level of collagen VI at the muscle basement membrane and mis-localization of the protein in interstitial and perivascular regions. CONCLUSIONS: The novel mutations we identified underscore the importance of THD and C2 domains in the assembly and function of collagen VI, thereby providing useful information for the genetic counseling of UCMD patients.

[Clinical and genetic characteristics of glucose transporter type 1 deficiency syndrome].

OBJECTIVE: To analyze the clinical and SLC2A1 gene mutation characteristics of glucose transporter type 1 deficiency syndrome. METHOD: The detailed clinical manifestations of six cases were recorded. The laboratory tests including EEG, MRI, blood chemistry, and lumbar puncture were performed. SLC2A1 gene mutations were analyzed by PCR, DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULT: Patient 1, 2 and 3 had classical clinical symptoms including infantile onset seizures, development delay. Patient 4, 5 and 6 had non-classical clinical symptoms including paroxysmal behavior disturbance, weakness, ataxia, lethargy, especially after fasting or exercise, without severe seizures. The plasma glucose levels were normal. The CSF glucose levels decreased in all the six cases, ranged from 1.10 mmol/L to 2.45 mmol/L, the mean level was 1.68 mmol/L. The CSF glucose/plasma glucose ratios decreased, ranged from 0.16 to 0.51, the mean ratio was 0.34. Four patients had normal EEG. Two patients had focal and diffuse epileptiform discharge, and one of them also had paroxysmal occipital or generalized high-amplitude slow waves during awake and sleep time. MRI abnormalities were found in three patients, patient 1 with mild brain atrophy, patient 3 with bilateral ventricle plump, and patient 4 with high signals in T2 in the frontal and occipital white matter, interpreted as hypomyelination. SLC2A1 gene mutations were found in six cases. Patient 1 has large scale deletion in exon 2. In patient 2 to 6, the mutations were c.741 G>A (E247K), 599delA, 761delA, c.1148 C>A (P383H), c.1198 C>T (R400C) respectively. Two patients were treated with ketogenic diet. The seizures disappeared and development became normal. Three patients responded to frequent meals with snacks. One patient refused any treatments, the symptoms continued to exist. CONCLUSION: The clinical manifestations of glucose transporter type 1 deficiency syndrome are varied. The common symptoms included infantile onset seizures and various paroxysmal events. These neurologic symptoms generally fluctuated and were influenced by factors such as fasting or fatigue. This feature could be a very important clue for the diagnosis of GLUT1-DS. Lumbar puncture is recommended in patients with episodic CNS symptoms especially after fasting. GLUT1-DS is a treatable neurometabolic disorder, early diagnosis and treatment may improve the prognosis of the patients.

[Clinical, pathological and molecular biological characteristics of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episode in children].

OBJECTIVE: To delineate the characteristics of the clinical manifestation, pathology of skeletal muscle and gene mutations of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episode (MELAS) in children. METHOD: The clinical manifestation, laboratorial data, brain images, muscle pathology and mitochondrial gene mutations were analyzed in 24 patients with MELAS who were diagnosed in Department of Pediatrics, Peking University First Hospital. Their prognosis was evaluated by following up. RESULT: Symptoms of central nervous system such as stroke-like episodes, vomiting, convulsion and headache were present in all the 24 cases. Nine cases had the symptoms of myopathy. Twenty cases had developmental delay. Short stature, being thin and hairy was very common in these cases. Serum lactate level increased in all the cases, pyruvate increased in 17 cases. Elevated CSF lactate was found in 2 cases. Magnetic resonance imaging (MRI) was performed on 24 cases, out of them 23 were abnormal. The lesions mainly involved cerebral lobes. Occipital lobe was the most common site of lesions. Computed tomography (CT) was performed on 13 cases, low density lesions were present in 10 cases, basal ganglia calcifications in 5 cases. Muscle biopsy was performed on 8 cases, ragged-red fibers (RRF) were found in 4/8 cases, and abnormal accumulation of mitochondria were found in 3/8 cases. The mtDNA gene mutational analysis showed A3243G mutation in these patients. The mutation rates varied from 11.6% to 75.0%. The same mutation were found in 4/5 mothers who had the genetic tests, and the mutation rates of the mothers varied from 15.0% to 23.6%. The clinical information of 11 cases was available through recent following up. Three cases died, the others had some degrees of mental retardation. CONCLUSION: Children with MELAS had various clinical manifestations. Central nervous system and skeletal muscle were usually involved. Short stature and hypertrichosis were common signs. The prognosis of this disease was disappointing. mtDNA A3243G was the most common mutation in MELAS. Fully understanding the characteristics of its clinical manifestation, laboratory tests, brain image, muscle pathology and molecular features can be helpful to the early diagnosis and treatment.

[Genetic and phenotypic characteristics of SCN1A mutations in Dravet syndrome].

OBJECTIVE: To study SCN1A gene mutations and their inheritance in patients with Dravet syndrome(DS), and to analyze the phenotypes of their family members and genotype-phenotype correlations. METHODS: Genomic DNA was extracted from peripheral blood samples from 181 DS patients and their parents. Phenotypes of affected members were analyzed. SCN1A gene mutations were screened using PCR-DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) RESULTS: SCN1A gene mutations were identified in 128 patients (70.7%), which included 60 missense mutations (46.9%), 55 truncation mutations (43.0%), 10 splice site mutations (7.8%), and 3 cases with SCN1A gene fragment deletions or duplications(2.3%). Five patients (3.9%) had mutations inherited from one of their parents. One father has carried a somatic mutation mosaicism (C373fsx378). For the 5 parents carrying a mutation, 1 had febrile seizures, 2 had febrile seizures plus, 1 had afebrile generalized tonic-clonic seizures, whilst 1 was normal. CONCLUSION: The mutation rate of SCN1A in DS patients is about 70%. Most mutations are of missense and truncation mutations. Only a few patients have carried fragment deletions or duplications. Most SCN1A mutations are de novo, only a few were inherited from the parents. SCN1A mutations carried by the parents can be in the form of mosaicism. The phenotypes of parents with SCN1A mutations are either mild or normal.

[Phenotype and SCN1A gene mutation screening in 39 families with generalized epilepsy with febrile seizures plus].

OBJECTIVE: To summarize the phenotypes and identify SCN1A mutations in families with generalized epilepsy with febrile seizures plus (GEFS(+)), and analyze the genotype- phenotype correlations in GEFS(+) families. METHOD: Genomic DNA was extracted from peripheral blood lymphocytes of the proband and other available members in the GEFS(+) families. The phenotypes of the affected members were analyzed. The coding regions and flanking intronic regions of the SCN1A gene were screened for mutations using PCR and direct DNA sequencing. RESULT: In 39 GEFS(+) families, there were 196 affected members, ranging from 2 to 22 affected members in each family. Their phenotypes included febrile seizures (FS) in 92(46.9%), febrile seizures plus (FS(+)) in 62(31.6%), FS or FS(+) with partial seizures in 12(6.1%), afebrile generalized tonic-clonic seizures (AGTCS) in 11(5.6%), myoclonic atonic epilepsy in 8(4.1%), Dravet syndrome in 2(1.0%), childhood absence epilepsy in 1 (0.5%), FS(+) with myoclonic seizures in 1(0.5%), AGTCS and myoclonic seizures in 1 (0.5%), partial seizures in 1 (0.5%), unclassified seizures in 5 (2.6%). Four families were found with SCN1A mutations, including three families with missense mutation (N935H, R101Q, G1382R) and one family with truncation mutation (C373fsx378). In three families with missense mutations, the phenotypes include FS, FS(+), FS(+) with partial seizures, and AGTCS. In one family with truncation mutation, the phenotypes included FS, FS(+), and Dravet syndrome. The mother of proband in the family with missense mutation (R101Q) and the father of proband in the family with truncation mutation (C373fsx378) were both somatic mosaicism. Both of their phenotypes were FS(+). CONCLUSION: The most common phenotypes of GEFS(+) were FS and FS(+), followed by the FS/FS(+) with partial seizures and AGTCS. The most severe phenotype was Dravet syndrome. SCN1A mutation rate in GEFS(+) was about 10%. Missense mutation was common in GEFS(+) families, few with truncation mutation. Few members of GEFS(+) families had somatic mosaicism of SCN1A mutations and their phenotypes were relatively mild.

[Clinical and genetic study of twelve Chinese patients with Alexander disease].

OBJECTIVE: To delineate the phenotype and genotype characteristics in 12 Chinese children with Alexander disease (AD), which is helpful for the molecular diagnosis and genetic counseling in China. METHODS: Clinical diagnosis of AD was based on MRI criteria proposed by van der Knaarp in 2001. Included AD patients were followed up for 0.50 - 3.67 years. Mutations in GFAP were detected by DNA sequencing. RESULTS: The 12 cases of AD were clinically diagnosed. Age of first visit was 4.87 years (0.75 - 12.00 years), with 3 types of chief complaints: developmental delay in 3, recurrent seizures in 7, unable to walk after falling in 2. Average head circumference was 52.34 cm (44 - 58 cm), which larger than age-matched average by 6.45% (1.80% - 13.95%). On the first visit, scaling according to Gross motor functional classification system (GMFCS) was performed, with GMFCSI in 8, II in 3, V in 1. Mild to severe cognitive dysfunction were found in 8, and seizures in 11 cases. The 12 patients were followed up for 0.50 - 3.67 years, their motor and cognitive function remained stable. Episodic aggravations provoked by fever or falling were observed in 5 cases (41.67%). Heterozygous missense mutations of GFAP were detected in 12 patients. All mutations were de novo; 3 out of 10 mutations identified were novel. R79 and R239 were hot mutations, which was consistent with previous reports. Mutations were located in exon 1 in 8 cases. CONCLUSIONS: The phenotype in these patients is characterized by slower progression compared with reports from other population and high incidence of seizures. And episodic aggravations provoked by fever or falling were more common. The genotype characteristics are consistent with previous reports. The results of this research expanded the number of patients with Alexander disease found to have GFAP coding mutations in China.

Genotype/phenotype of 6 Chinese cases with Niemann-Pick disease type C.

UNLABELLED: Niemann-Pick disease type C (NP-C), caused by mutations of either NPC1 or NPC2 gene, is an inherited lysosomal lipid storage disorder that is difficult to be diagnosed and treated. NP-C is rarely reported in China and so far very few literatures are available for Chinese clinical workers. To better characterize this disease in China and improve genetic counseling, mutational analyses of NPC1 gene were carried out in 6 unrelated Chinese patients. METHODS: Clinical data of the probands from 2007 to 2010 were collected and analyzed. All exons of NPC1 were analyzed by direct sequencing. RESULTS: The six cases, four males and two females, included three cases of late infantile subtype and three cases of juvenile subtype. Case one and case six had siblings who suffered from the same disease. The onset of clinical symptoms varied from three to ten years old, and they included progressive cognitive and language impairment, and motion retrogradation. All were caught by focal or generalized seizures from one to four years after the onset. Vertical supranuclear gaze palsy, dysarthria, dysphagia, internal rotation and adduction of bilateral hands and splenomegaly occurred gradually during the disease progression. Five patients had laughter-cataplexy. MRI indicated mild brain atrophy. Sea blue cells and Niemann-Pick cells were presented in bone marrow smears. Activity of acid sphingomyelinase was normal or only slightly lower than controls. Supporting and symptomatic treatments could improve some of the clinical signs. We identified 10 different NPC1 mutations were identified in 12/12 alleles, 3 of which are described for the first time. All mutations were missense mutations, which located throughout the gene with five clustering in the cysteine-rich luminal domain. Homozygous mutation of S865L correlated with a relatively severe juvenile neurological form. CONCLUSIONS: NP-C is a rare autosomal recessive lysosomal storage disease that affects intellectual development of children, causing dementia, vegetative state and eventual death. The awareness of NP-C should be raised in the Chinese population. The typical clinical features of this disease include vertical supranuclear gaze palsy, seizures and cataplexy. Laboratory features include the presence of sea blue cells and Niemann-Pick cells in bone marrow smears. NPC1 mutation can be identified in most of these patients and most of them are missense mutations.

[Analysis of HEPACAM mutations in a Chinese family with megalencephalic leukoencephalopathy with subcortical cysts].

OBJECTIVE: To explore HEPACAM mutations in a Chinese family with megalencephalic leukoencephaloptathy with subcortical cysts (MLC). METHOD: Genomic DNA samples were extracted from peripheral blood of the proband and her parents. All exons and exon-intron boundaries of HEPACAM and MLC1 were amplified in the MLC family by polymerase chain reaction (PCR) followed by direct DNA sequencing. RESULT: Two heterozygous mutations of HEPACAM located in exon 2, c.203A > T(p.K68M) and c.395C > A(p.T132N), were identified in the proband. The proband's mother had the heterozygous mutations c.203A > T(p.K68M), and her father had the heterozygous mutation-c.395C > A(p.T132N). There was no variation found in MLC1 gene. CONCLUSION: The proband was heterozygous compound MLC patient carrying on one allele with the c.203A > T(p.K68M) mutation inherited from her mother, and the other allele with the c.395C > A(p.T132N) mutation inherited from her father. The parents both are heterozygous carriers with normal phenotype. The disease-causing gene for this family was resulted in HEPACAM mutation other than MLC1 mutation.

Niemann-Pick disease type C: analysis of 7 patients.

BACKGROUND: Niemann-Pick disease type C (NP-C), derived from mutation of the NPC1 or NPC2 gene, is one of the recessive lysosomal lipid storage disorders that are difficult to diagnose and treat. Since NP-C has been rarely reported in China, we reviewed 7 patients with NP-C. METHODS: The 7 patients had been diagnosed with NP-C from 2007 to 2010 at our department and their laboratory and clinical data were analyzed. RESULTS: The 7 patients, 5 males and 2 females, included 4 patients of late infantile subtype and 3 patients of juvenile subtype, in which patients 2 and 3 were siblings. Their clinical symptoms occurred from 4 to 10 years of age, exhibiting as progressive cognitive and language impairment as well as motor retrogression. Six patients were caught by focal or generalized seizures from 1 to 4 years after the onset of the disease. Vertical supranuclear gaze palsy, dysarthria, dysphagia, internal rotation and adduction of bilateral hands and splenomegaly occurred following the progress of clinical symptoms. Five patients had laughter-cataplexy. MRI showed mild brain atrophy in 6 patients. Reduction of total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol occurred in 6 patients. Sea-blue cells and Niemann-Pick cells were found in bone marrow smears. The activity of acid sphingomyelin enzyme was normal or only slightly lower. Supporting or symptomatic treatment improved common clinical symptoms. CONCLUSIONS: NP-C is a rare autosomal recessive inherited lysosomal storage disease that affects the intellectual development of children and may lead to dementia, vegetative state or death. Clinical features of this disease include vertical supranuclear gaze palsy, seizures and cataplexy. Laboratory features include abnormal plasma cholesterol level, and sea-blue cells and Niemann-Pick cells in bone marrow smears. The treatments of the disease include supporting or symptomatic administration.

Long-term effects of recurrent neonatal seizures on neurobehavioral function and related gene expression and its intervention by inhibitor of cathepsin B.

Cathepsins are families of proteases that have been reported to play the key roles in neuroexcitotoxicity. The present study was sought to determine the effect of CBI, a cathepsin B inhibitor, in the prevention of neurobehavioral deficits after inhalant flurothyl-induced recurrent neonatal seizures in rats. We examined the expression pattern of autophagy-related genes at acute phase after the last seizures using western blot method, and evaluated behavioral deficits during postnatal day 28 (P28) to P35. The results showed improved neurological scores and learning ability in CBI-treated rats compared with the nontreated control. Flurothyl-induced increases in the ratio of LC3-II/LC3-I, Beclin-1 and Cathepsin-B were blocked by pre-treatment with CBI at 1.5, 3, 6 and 24 h after the last seizures in hippocampus and cerebral cortex by western blot analysis. Meanwhile, CBI also reversed flurothyl-induced down-regulation of Bcl-2 protein levels. Furthermore, in the long-term time point of 35 days (P35), PRG-1 mRNA and protein level in hippocampus and cerebral cortex of recurrent seizure group were up-regulated when compared to the control rats; meanwhile, the up-regulated expression of PRG-1 were robustly inhibited by CBI. These date demonstrated, for the first time, that lysosomal enzymes participate in neonatal seizure-induced brain damage and that modulation of cathepsin B may offer a new strategy for the development of therapeutic interventions for treatment of developmental seizure-induced brain damage.

[Electroclinical features of myoclonic-atonic epilepsy].

OBJECTIVE: To summarize the electroclinical characteristics of myoclonic atonic epilepsy (MAE) in children. METHOD: The clinical data, video electroencephalogram (EEG) and simultaneous electromyography (EMG) of MAE patients were analyzed. The treatment and its effects were followed up. RESULT: In 47 MAE patients, 25 had a history of febrile seizures (FS), 20 had a family history of FS or epilepsy. All patients had a normal development before the illness. The age of afebrile seizure onset was between 1.4 years to 5.8 years. The first seizure was generalized tonic-clonic seizure (GTCS) in 41 patients (87.2%). All patients had multiple seizure types, including 47 GTCS (97.9%), 34 myoclonic atonic seizures (72.3%), 47 myoclonic seizures (100%), 32 atonic seizures (68.1%), 36 atypical absences (76.6%) and 3 tonic seizures (6.4%). EEG backgrounds were slow or parietal θ rhythm, interictal EEG showed 1-4 Hz (predominant 2-3 Hz) generalized spike and wave or poly spike and wave discharges in all cases. Seizures were controlled by antiepileptic drugs (AEDs) in 41 patients (87.2%). Valproate was used in 37. Lamotrigine was used in 26. Mild mental retardation was observed in 10 children after the onset of the illness. CONCLUSION: The clinical features of MAE included the following: the development was normal before the onset of the illness; the onset of seizure type was often GTCS. All patients had multiple generalized seizure types. Myoclonic atonic seizure was its characteristic seizure type. EEG showed generalized discharges. Early diagnosis and rational choice of AEDs are important for getting a better prognosis.

[Clinical and mutation analysis of a Chinese family with muscle eye brain disease].

OBJECTIVE: To study the clinical feature of a Chinese family with muscle-eye-brain disease (MEB) and the mutation of protein O-linked-mannose beta-1, 2-N-acetylglucosaminyltransferase 1 gene (POMGNT1). METHODS: Clinical data of the proband and his family members were collected. Genomic DNA from the patient and his parents was extracted using standard procedures from the peripheral blood leukocytes. Polymerase chain reaction and DNA direct sequencing were employed to analyze all of the exons to determine the mutation, and the relationship between genotype and phenotype was analyzed. RESULTS: The proband was diagnosed as floppy baby, presented with delayed psychomotor development and myopathic face. His serum creatine kinase (CK) level elevated moderately and brain MRI showed cerebral and cerebellar gyrus abnormalities with white matter signal intensity changes, cerebellar cysts and cerebellar and brain stem hypoplasia, consistent with congenital muscular dystrophy with eye brain disorder. Further test with DNA detected a compound heterozygous mutation of c.1896 1 G to C before exon 22 which may induce splicing error, and missense mutation c.1319T to G, p.L440R in exon 16. Both parents had a heterozygous mutation at the mutation sites. CONCLUSION: According to our study, the family is diagnosed as MEB. The proband carried compound heterozygous mutations in the POMGNT1 gene, and his parents are heterozygous carriers, which is consistent with autosomal recessive inheritance. The child is definitely diagnosed as having muscle eye brain disease.

[Association of genetic polymorphisms in methionine metabolism genes with X-linked adrenoleukodystrophy].

OBJECTIVE: To investigate the association of the polymorphisms of methionine metabolism genes and the phenotype of X-linked adrenoleukodystrophy (X-ALD) and clinical severity. METHODS: The clinical information of 120 X-ALD patients were analyzed and three genetic variants involved in the methionine metabolism, including cystathionine beta-synthase (CBS) c.844_855ins68, 5-methyltetrahydrofolate-homocysteine-S-methyltransferase (MTR) c.2756A to G, and transcobalamin 2 (TC2) c.776 C to G were analyzed by polymerase chain reaction and sequencing. The association between these polymorphisms and phenotype of X-ALD was studied. RESULTS: The frequency of GG genotype of the TC2 c.776 C/G was higher in patients with central nervous system(CNS) demyelination than in controls (P= 0.012). However, the other two polymorphisms did not show any significant associations with the phenotypes. CONCLUSION: The GG genotype of TC2 c.776 C/G may contribute to X-ALD phenotype.

[Clinical characteristics and follow-up management of 135 children with myasthenia gravis].

OBJECTIVE: To investigate and analyze the clinical manifestations, classification, therapeutic approaches and follow-up of myasthenia gravis (MG) in children in order to improve its management and prognosis. METHODS: Clinical information of 135 children with MG, who were diagnosed between January 1993 to January 2008, were collected and retrospectively analyzed. And prospective following-up of these patients were conducted. RESULTS: Among the 135 cases, 59 were males and 76 females, giving the ratio of M/F around 1:1.3. Totally, 115 cases (85.2%) were type I MG (ocular type), of which only 4.2% developed to generalized type during the subsequent clinical course. Type II MG (generalized type)was found in 18 cases (13.4%) and type III MG in two cases(1.5%). The onset age ranged from 5 month to 15 years, with 50.3% before three years and 80.7% before seven years. Upper respiratory tract infection was presented in 26.7% (36/135) of the sick children before the onset of MG. Among the 106 children being followed up, recurrence of the disease identified in 50.9% and the number of relapse ranged from 1 to 9. Altogether, 40.19% (43/106) of the cases were positive for anti-acetylcholine receptor antibodies (AchR-Ab) on the initial examination, and the AchR-Ab postitive rate showed no difference among different clinical subtypes and states. However, during the follow-up, 53% (9/17) of the recurrent cases, who were negative at the first onset, turned to be positive, and 37.97% (30/79) were positive for repetitive nerve stimulation in electromyogram test. There were 71 % (45/63) of all the cases showed reduced levels of CD4+ and/or CD3+ and/or CD8+. Thymus proliferation was found in 5.93% (8/135) through CT scan and thymoma in 1.48% (2/135). Steroids and anti-cholinesterase administration were effective in most cases with good prognosis. CONCLUSION: Childhood MG, mainly type I, is relatively common in China, with specific characteristics which are different from western patients or adult MG in morbidity, sex distribution, progress, laboratory examination and treatment. The prevalence of myasthenia gravis crisis and mortality rate in MG children is low, and few are accompanied with thymoma. Most MG cases have a satisfied prognosis and few have neuropsychic sequela.

[Clinical characteristics of Huntington disease in two pedigrees and analysis of expanded CAG trinucleotide repeat].

OBJECTIVE: To understand the clinical and genetic features of Huntington disease (HD). METHODS: The clinical data of HD cases from 2 Chinese families were analyzed and trinucleotide repeat in the IT15 gene were investigated in 9 of the two families by polymerase chain reaction and GeneScan. RESULTS: Among the two pedigrees, 6 cases were ascertained as HD by genetic test. Genotypes of IT15 were heterozygous in these HD patients. CAG repeat of the patients in the HD chromosome were 40-78. In the two pedigrees, the onset age was earlier in the subsequent generations than that of their fathers. In pedigree 2, the onset age was inversely correlated with CAG repeat number. One out of the 6 cases was juvenile-onset type of Huntington disease, whose clinical symptoms were different from those of the adult-onset cases, especially the hypertonic manifestation. CONCLUSION: HD is an autosomal dominant neurodegenerative disorder with genetic anticipation caused by enlargement of CAG repeat in IT15 gene. The clinical manifestation is different between the juvenile-onset and the adult-onset. The number of CAG repeat is inversely correlated with the onset age and clinical severity.

Acute phase expression pattern of ZnTs, LC3, and beclin-1 in rat Hippocampus and its regulation by 3-methyladenine following recurrent neonatal seizures.

It has been reported that autophagy and zinc transporters (ZnTs) both play the key roles in excitotoxicity, which is associated with cognitive deficits following developmental seizures. However, the influence of autophagy on acute phase ZnTs expression has never been studied. The present study sought to investigate the contribution of an autophagy inhibitor (3-methyladenine, 3-MA) on the regulation of ZnTs, microtubule-associated protein 1A/1B light chain 3 (LC3), and beclin-1 expression in rat hippocampus following recurrent neonatal seizures. We examined the expression of ZnT1~ZnT3, LC3, and beclin-1 at 1.5, 3, 6, and 24 h after the last seizures using real-time RT-PCR and Western blot methods, respectively. The results showed that there were upregulated expressions of ZnT-1, ZnT-2, LC3, and beclin-1 of RS group. Pretreatment with 3-MA remarkably attenuated seizure-induced ZnT-1, ZnT-2, LC3, and beclin-1 increase. Additionally, linear correlations could be observed between LC3-Beclin1, LC3-ZnT-2, Beclin1-ZnT2, Beclin1-ZnT3, and among ZnT1~ZnT3 in control group, while the linear correlations could be observed between LC3-Beclin1, Beclin1-ZnT2, and Beclin1-ZnT3 in RS group. These results demonstrate, for the first time, that there exists an interaction of Zn(2+) with autophagic signals that are immediately activated in hippocampus after recurrent neonatal seizures, which might play a key role in neonatal seizure-induced excitotoxicity.

[Genotype, phenotype analysis and follow-up study on patients with Duchenne/Becker muscular dystrophy].

OBJECTIVE: To improve the diagnosis and management of Duchenne/Becker muscular dystrophy(DMD/BMD). METHODS: Clinical features of 90 cases of DMD/BMD were collected. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes, and multiplex ligation-dependent probe amplification (MLPA) was applied to detect DMD gene to identify genetic mutation. For those patients whose deletion/duplication mutation was not identified, FKRP gene mutation analysis was performed using PCR-DNA direct sequence. All the cases were followed up. RESULTS: Among the 90 cases of clinically diagnosed DMD/BMD, exons deletion of DMD was detected in 58 cases (64.44%), and exons duplication in 9 (10.00%). Among the 34 mothers with an affected boy but without previous genetic conformation, 17 were confirmed to be carriers with gene deletion/duplication. None of the 23 cases, without detected DMD gene deletion/duplication, carried FKRP gene mutation. Fourteen children were given short-term intermittent prednisone therapy (0.75 mg/kg daily during the first 10 days of each month). The course was not long enough and the sample size was too small to conclude any benefits or side effects. Prenatal diagnosis was provided for one mother in her next pregnancy detecting a female carrier fetus. CONCLUSION: DMD gene deletions mainly occurs between exons 45 and 54, while duplications mostly at 5'-terminus. Identification of the characteristics and types of gene mutation may facilitate the recognition and prognosis prediction of DMD/BMD. MLPA is a non-complex and quick diagnostic tool for DMD/BMD and its carriers, and also helpful in genetic counseling.

[Analysis of the parental origin of MECP2 mutations in patients with Rett syndrome].

OBJECTIVE: To identify the parental origin of methyl-CpG-binding protein 2 (MECP2) gene mutations in Chinese patients with Rett syndrome. METHODS: Single nucleotide polymorphisms (SNPs) in intron 3 of the MECP2 gene were analyzed by PCR and sequencing in 115 patients with Rett syndrome. Then sequencing of the SNP region was performed for the fathers of the patients who had at least one SNP, to determine which allele was from the father. Then allele-specific PCR was performed and the products were sequenced to see whether the allele from father or mother harbored the mutation. RESULTS: Seventy-six of the 115 patients had at least one SNP. Three hot SNPs were found in these patients. They were: IVS3+22C >G, IVS3+266C >T and IVS3+683C>T. Among the 76 cases, 73 had a paternal origin of MECP2 mutations, and the other 3 had a maternal origin. There were multiple types of MECP2 mutation of the paternal origin, including 4 frame shift, 2 deletion and 67 point (56C >T, 6C >G, 2A >G, 2G >T and 1A >T) mutations. The mutation types of the 3 patients with maternal origin included 2 frame shift and 1 point (C >T) mutation. CONCLUSION: In Chinese RTT patients, the MECP2 mutations are mostly of paternal origin.

Dynamic pattern of gene expression of ZnT-1, ZnT-3 and PRG-1 in rat brain following flurothyl-induced recurrent neonatal seizures.

Zinc transporters (ZnTs) and plasticity-related genes (PRGs) both play the key roles in the formation of hippocampal mossy fiber sprouting, which is associated with cognitive deficits following developmental seizures. Here, for the first time, we report the timing of expression pattern of ZnT-1, ZnT-3 and PRG-1 in hippocampus and cerebral cortex following developmental seizures. A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6). Rats were assigned into the recurrent-seizure group (RS, seizures induced in 6 consecutive days) and the control group. At 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h, 7 d and 14 d after the last seizures, the mRNA level was detected using RT-PCR method; PRG-1 protein level was examined by Western blotting analysis. At an early period of 12 h and 48 h after the last seizures, both ZnT-1 and ZnT-3 showed significantly down-regulated mRNA level in the cerebral cortex of RS group than those at the corresponding time point in control group. In the long-term time point of 14 d after the last seizure, ZnT-3 mRNA and PRG-1 protein level in hippocampus were up-regulated while the mRNA level of ZnT-1 down-regulated; in addition, there were up-regulated level of both the mRNA and protein level of PRG-1 and down-regulated mRNA level of ZnT-3 in the cerebral cortex of RS group when compared to the control. Taken together, these dates are consistent with an important role for ZnT-1, ZnT-3 and PRG-1 in the pathophysiology of the long-term adverse effects of recurrent neonatal seizure-induced hippocampal mossy fiber sprouting and cognitive deficit.