[Paroxysmal crying and motor regression for more than two months in an infant].

The patient was a male who was found to be abnormal at the age of 4.5 months. He presented with irritability, motor regression and opisthotonus. Brain MRI revealed bilateral abnormality in the lentiform nucleus, thalamus, deutocerebrum and cerebellar hemispheres. Novel compound heterozygous mutations of SLC19A3 gene, c.950G>A(p.G317E) and c.962C>T(p.A321V), were found in the patient. Further study showed that c.950G>A was inherited from his father and c.962C>T came from his mother. Using bioinformatics software analysis, both of the mutations were found to be harmful. His symptoms were improved remarkably after biotin, thiamine and "cocktail" therapy. One month later a brain MRI revealed that the lesions in basal ganglia and cerebellar hemispheres were improved. The patient was definitely diagnosed with biotin-thiamine responsive basal ganglia disease (BTBGD). BTBGD is a treatable autosomal recessive disease and early administration of biotin and thiamine may lead to clinical improvement.

[Myoclonus epilepsy with ragged-red fibers: a case report and literature review].

To demonstrate the clinical manifestation, diagnosis and treatment of myoclonus epilepsy with ragged-red-fibers (MERRF), a case of MERRF was presented with review of the literature. A 4-year-7-month-old girl was diagnosed with MERRF. She had tremor, fatigue and developmental delay for more than 2 years. Laboratory tests showed that the serum and urine lactic acid and pyruvic acid increased significantly. Electroencephalogram showed diffuse and focal spike slow wave and slow wave in right central and parietal regions. Electromyogram showed neurological damage. Gene mutational analysis showed mtDNA 8344 A>G mutation. The mutational rate was 78%. Mitochondrial disease MERRF syndrome was diagnosed. Cocktails therapy with vitamins B1, B6, B12, L-carnitine, and coenzyme Q10 was administrated to the patient. MERRF is a rare disease. The diagnosis can be made by gene mutational analysis. Cocktail therapy may slow down the deterioration of the disease. Gene therapy is still experimental.

[Long-term outcome and related factors of epileptic seizures in children].

OBJECTIVE: To analyze the Long-term outcome of seizures, and to explore the effects of related factors, including the age at onset, types of epileptic syndromes, and etiological factors, etc. METHODS: The clinical data were retrospectively surveyed from 265 children with regular follow-ups for over 1 year at Peking University First Hospital (Jan. 2003 to Dec. 2006). The seizure-free rate was calculated as an at least one-year non-occurrence of seizures. The Long-term outcome of seizures was analyzed in association with factors including the age at onset, types of epileptic syndromes, and etiology. RESULTS: (1) Seizure types were clarified in all the cases, with combined types of seizures in 17. Epileptic syndromes were identified in 163/265 cases (61.5%). With regular antiepileptic drug therapy, 57.9% children with epilepsy could be seizure-free. (2) Seizure-free was demonstrated in 142/265 cases with a seizure-free rate of 53.6% in this group. (3) The age at onset was youngest in the non-efficacy group. (4) The seizure-free rate was different by syndrome types of epilepsies, with a higher seizure-free rate in idiopathic generalized epilepsy (72.4%) and benign epilepsy in children with centro-temporal spikes (65.5%), whereas a lowest rate (21.7%) in infantile spasms. (5) A significant difference of seizure-free rates was revealed in different etiological groups. Children with idiopathic epilepsy achieved higher seizure-free rate (69.2%) than those with symptomatic and cryptogenic epilepsy (45.4%). CONCLUSION: The epilepsy children with regular antiepileptic drug therapy had generally satisfactory outcome of seizures, with over half cases of seizure-free. The prognosis was demonstrated to be closely related with the etiological factors, syndrome types and age at onset.

[STXBP1 gene mutation in newborns with refractory seizures].

OBJECTIVE: To study the relationship between STXBP1 gene mutations and refractory seizures with unknown causes in newborns. METHODS: The coding region of STXBP1 gene was detected using direct Sanger sequencing in 11 newborns with refractory seizures of unknown causes. RESULTS: STXBP1 gene mutation was found in 1 out of 11 patients. It was a missense mutation: c.1439C>T (p.P480L). CONCLUSIONS: STXBP1 gene mutation can be found in neonatal refractory seizures of unknown causes, suggesting a new approach of further research of this disease.

Barriers to diagnosis of a rare neurological disorder in China--lived experiences of Rett syndrome families.

Rett syndrome is a rare neurological disorder affecting girls and usually caused by a mutation on the MECP2 gene. It is estimated that approximately 1,000 girls are born every year in China with Rett syndrome but far fewer have received a diagnosis. Fourteen of 74 Chinese families known to the International Rett Syndrome Phenotype Database participated in this qualitative study. Telephone interviews were conducted in Mandarin to explore pathways to a diagnosis of Rett syndrome in China and associated barriers. Families consulted multiple clinical centers and eventually received a diagnosis at a centrally located hospital. Over the course of this pathway, families encountered lack of knowledge and diagnostic expertise for Rett syndrome at local levels and a heavily over-burdened hospital system. There was a paucity of information available to guide management of this rare disorder after the diagnosis had been received. Our study suggests that the frustrations experienced by families could in part be addressed by the provision of information, education, and training related to Rett syndrome for clinicians, additional resources to allow clinicians to request genetic testing for confirmation of the clinical diagnosis and for information and support services for families.

[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.

[Value of latex agglutination test in the diagnosis and treatment of cryptococcal meningitis in children].

OBJECTIVE: To evaluate the value of cryptococcal latex agglutination test in the diagnosis and treatment of cryptococcal meningitis in children. METHODS: The clinical data of 10 children with cryptococcal meningitis were retrospectively studied. Cryptococcal meningitis was confirmed based on clinical manifestations, India ink stain, cryptococcal latex agglutination test or cryptococcal culture. The outcome of antifungal treatment and the changes of latex agglutination test titer were followed up for 2 to 4 years. RESULTS: Latex agglutination test and/or India ink stain were positive (titer 1 : 64-1 : 1024) in 8 patients in the first examination of cerebrospinal fluid. In the other 2 patients, latex agglutination test was positive (titer 1 : 256) in the fourth examination of cerebrospinal fluid in one, and India ink stain was positive in the eleventh examination in the other. After antifungal treatment, six patients were cured, two patients died, and two patients were lost to follow-up. The positive cryptococcal latex agglutination test (titer 1 : 2-1 : 16) was seen respectively in six, three, two and one cured patients 6 months, 1 year, 2 years and 4 years later. CONCLUSIONS: The cryptococcal latex agglutination test of cerebrospinal fluid is valuable for the quick and early diagnosis of cryptococcal meningitis; however, the decision of withdrawal of antifungal treatment should not rely on the results of the test.

[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.

[Research progress of Rett syndrome causing gene MECP2--the structure, function and modulation of MECP2].

Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder characterized by regression of language, stereotype hand movement and loss of purposeful hand use, is primarily caused by mutation of menthyl-CpG-binding protein 2 (MECP2). The 76 kb human MECP2 is characterized by three salient features: a very large intron 2 (60 kb), an 8.5 kb 3'-UTR with highly conserved regions and different polyadenylation sites, and a 40 kb intergenic region separating MECP2 from the nearest upstream gene. There are two isoforms of MeCP2, MeCP2e1 and MeCP2e2. The differences between the two isoforms, the function of the 3'-UTR and the long-range cis-regulatory sequences in the intergenic region were extensively studied. In contrast to initial report, recent studies show that MeCP2 binds not only to methylated promoters and silence transcription, but also to the sites outside of genes containing only a few of CpG islands. Furthermore, MeCP2 can function as both an activator and a repressor of transcription.

[Methyl-CpG-binding protein 2 gene and CDKL5 gene mutation in patients with Rett syndrome: analysis of 177 Chinese pediatric patients].

OBJECTIVE: To study the spectrum of mutations in methyl-CpG-binding protein 2 gene (MECP2) and cyclin-dependent kinase-like 5 gene (CDKL5) in Chinese pediatric patients with Rett syndrome (RTT), and establish a simple, quick, and efficient gene test method as well as screen a strategy of genetic diagnosis for RTT. METHODS: Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes of 117 pediatric patients diagnosed from 1987 to 2007. PCR was used to amplify the exons 1 - 4 of MECP2 using published primers. If no mutation was identified after screening exons 2 - 4, exon 1 was screened. If no mutation was identified in MECP2 by sequencing, multiplex ligation dependent probe amplification (MLPA) was employed to screen for large deletions by using P015C kit. If no mutation was identified in the MECP2 by sequencing and MLPA respectively, then the coding region of CDKL5 was screened by denaturing high performance liquid chromatography (DHPLC). RESULTS: The total mutation frequency in MECP2 and CDKL5 genes among all RTT patients was 82%. MECP2 mutations were found in 86% (137/159) of the patients with classical RTT and in 44% (8/18) of those with atypical RTT. Most of the mutations were missense mutations, accounting for 39%, followed in order of frequency by nonsense mutations 28%, frame shift mutations 17% and large deletions 14.5%. The eight most frequent MECP2 mutations were p.T158M (13%), p.R168X (12%), c.806delG (7%), p.R255X (6%), p.R270X (5%), p.R133C (5%), p.R306C (4%), and p.R106W (3%), with p.T158M as the most common of the MECP2 mutations and c.806delG as a hotspot mutation in Chinese patients with RTT. Only one synonymous mutation was identified in CDKL5. CONCLUSION: The spectrum of MECP2 mutations within the mainland Chinese RTT patients is similar to that of those patients reported in the world. p.T158M, p.R168X, c.806delG, p.R255X, p.R270X, p.R133C, p.R306C, and p.R106W are the hotspot mutations of MECP2 and c.806delG is a specific hotspot mutation in Chinese patients with RTT. The most effective method to screen mutations is to screen the exon 4. MLPA is an effective supplement to the routine methods.

[Multiplex ligation-dependent probe amplification analysis of subtelomeric chromosome rearrangements in children with idiopathic mental retardation].

OBJECTIVE: To study the rate of subtelomeric rearrangements in patients with idiopathic mental retardation (MR) and to search the cause of MR. METHODS: DNA was extracted and purified from peripheral blood leukocytes of 180 patients with idiopathic MR. DNA was tested using specific subtelomeric multiplex ligation-dependent probe amplification (MLPA) kits P036B/C and P070 according to manufacturer's instructions. The amplification products were separated by capillary electrophoresis using an ABI 3100 automated sequencer and size standard. MLPA data were extracted by GeneScan Analysis software. Data normalization and analysis were performed with the built-in MLPA application in GeneMarker. RESULTS: Among 180 patients with idiopathic MR, 12 had pathological subtelomeric deletions including 3 cases with a 4p deletion, 2 cases with a deletion at 9q and 22q respectively, 1 case with a deletion at 1p, 7p, 8p, 9q and 12q respectively. Subtelomeric rearrangements were responsible for 7% cases of idiopathic mental retardation. CONCLUSION: Subtelomeric rearrangement is a common cause of idiopathic mental retardation.

Levetiracetam administration is correlated with lower mortality in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes: a retrospective study.

BACKGROUND: Studies on the relationship between antiepileptic drug (AED) administration and clinical outcomes in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) remain scarce. Levetiracetam (LEV) is an AED that is neuroprotective in various neurologic disorders. This study aimed to determine the impact of LEV on the outcome of MELAS. METHODS: A retrospective, single-center study was performed based on a large cohort of patients with MELAS with a history of seizures (n = 102). Decisions on antiepileptic therapies were made empirically. Patients were followed up for 1 to 8 years (median, 4 years) and divided into 2 groups based on whether LEV was administered (LEV or non-LEV). The modified Rankin scale (mRS) scores and mortality risks were analyzed in all patients. RESULTS: LEV, carbamazepine, benzodiazepines, topiramate, oxcarbazepine, valproate, and lamotrigine were administered in 48, 37, 18, 13, 11, 9, and 9 patients, singly or in combination, respectively. The mean mRS score of the LEV group (n = 48) was lower than that of the non-LEV group (n = 54; mean ±â€Šstandard deviation, 2.79 ±â€Š1.47 vs. 3.83 ±â€Š1.93, P = 0.006) up to the end of the study. Nevertheless, there was no difference in the proportion of subjects without disability (mRS ranging 0-1) between the groups (P = 0.37). The multivariate regressions revealed that LEV treatment was associated with lower mRS scores (odds ratio 0.32, 95% confidence interval [CI] 0.15-0.68, P = 0.003) and mortality rates (hazard ratio 0.24, 95% CI 0.08-0.74, P = 0.013). There was a significant difference in the Kaplan-Meier survival curves between the groups (χ = 4.29, P = 0.04). CONCLUSIONS: The LEV administration is associated with lower mortality in patients with MELAS in this retrospective study. Further laboratory research and prospective cohort studies are needed to confirm whether LEV has neuroprotective effects on patients with mitochondrial diseases.

[Methylation-specific multiplex ligation-dependent probe amplification in diagnosis of Prader-Willi syndrome and Angelman syndrome].

OBJECTIVE: To verify the sensitivity and reliability of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and to develop a simple, accurate, reliability method of genetic diagnosis for AS and PWS. METHODS: Peripheral blood samples were collected from 4 suspected AS patients, 2 suspected PWS patients, 2 normal persons, and 2 molecular biologically proven positive controls (1 AS patient and 1 PWS patient). DNA was extracted and purified. MS-MLPA was used to detect the methylation of the CpG dinucleotide and the copy number in the 15q-q13 region. The results of MS-MLPA were confirmed by MSP. RESULTS: Three cases with maternal deletion on 15q11-q13 region and one case with paternal uniparental disomy (UPD) or imprinting center defect in 15q11-q13 region were found in the 4 suspected AS patients. One PWS case was found to be with paternal deletion in 15q11-q13 region and the other with paternal deletion in 15q11-q13 region or UPD or imprinting center defect in 15q11-q13 region. CONCLUSION: MS-MLPA is a simple, rapid, accurate, and reliable method of genetic test.

[Prenatal diagnosis of X-linked adrenoleukodystrophy].

OBJECTIVE: To make prenatal dignosis of X-linked adrenoleukodystrophy (ALD) for the prevention of the disease. METHODS: Eighteen amniocenteses were performed on 17 suspected carriers of X-ALD during 18-30 gestation weeks. The very long chain fatty acids (VLCFAs) levels of cultured amniocytes were tested by gas chromatography-mass spectrometry (GC/MS). The plasma VLCFAs levels were measured in 8 of the 18 prenatal diagnosed children when they were born or after abortion. ABCD1 gene mutation analysis was carried out in 8 cases by PCR and sequencing. ALDP of amniocytes was tested by Western blotting in 2 cases from a family, one female, another male, and the VLCFAs of cultured amniocytes were increased in both of them. RESULTS: Among the 18 fetuses, 10 were males and 8 were females. The VLCFAs levels of the cultured amniocytes were increased in 3 males and 4 females. The postnatal plasma VLCFAs were normal in 5 cases with normal VLCFAs levels of amniocytes, and increased in 3 cases with high VLCFAs levels of amniocytes. ABCD1 gene mutations were found in 4 cases with high VLCFAs levels of amniocytes, no mutation was found in other 4 cases with normal VLCFAs levels of amniocytes. ALDP of amniocytes could be detected in the female with high VLCFAs levels of amniocytes, and it could not be detected in the male with high VLCFAs levels of amniocytes. Three male fetuses with high VLCFAs levels of amniocytes were aborted. The others who were born were normal clinically so far. CONCLUSION: The prenatal diagnosis is very important for the prevention of ALD. Amniocyte VLCFAs level analysis combined with ABCD1 gene mutation analysis and ALDP test could make a proper prenatal diagnosis.

Clinical and laboratory survey of 65 Chinese patients with Leigh syndrome.

BACKGROUND: Leigh syndrome is an inherited neurodegenerative disease that emerges in infancy and childhood and presents with a clinically heterogeneous variety of neuromuscular and non-neuromuscular disorders. It can result from the inheritance of mutations in either nuclear or mitochondrial DNA. In the current study, we performed a retrospective study in 65 patients in order to investigate the clinical and genetic characteristics of Leigh syndrome in Chinese patients. METHODS: Sixty-five unrelated cases (35 men and 30 women) who were hospitalized in the past 12 years were reviewed. Diagnosis was based on both the clinical presentation and the characteristic neuropathologic findings of bilateral symmetric necrotizing lesions in the basal ganglia and brain stem as detected using cranial computed tomography (CT) scan or magnetic resonance imaging (MRI). The differential diagnosis of organic acidurias and fatty acid beta-oxidation defects were performed. Specific point mutations and deletions in mitochondrial DNA (T8993G, T8993C, T9176C, A8344G, A3243G) were screened by PCR-restriction analysis and Southern blot. The SURF1 gene was sequenced. Skeletal muscle biopsies were performed in 17 (26.2%) of the patients. The diagnosis was confirmed by autopsy in 6 (9.2%) patients. RESULTS: The patients had various forms of metabolic encephalomyopathy. Fifty-nine (90.8%) of the patients had the typical neuroradiological features of Leigh syndrome, including symmetrical necrotizing lesions scattered within the basal ganglia, thalamus and brain stem. Twenty (30.8%) patients were confirmed by genetic, biochemical analysis and autopsy. Specific point mutations in mitochondrial DNA were found in 5 cases (7.7%). Of these, the A8344G mutation was detected in 2 patients. The T8993G, T8993C, and A3243G point mutations were identified in 3 other patients, respectively. SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 (12.3%) families by DNA sequencing. A G604C mutation was identified in 6 (9.2%) patients. The genotypes of 52 patients remained unknown. CONCLUSIONS: Leigh syndrome presents as a diverse array of clinical features and can result from specific mutations in nuclear or mitochondrial DNA. In this study, SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 (12.3%) out of 65 patients with Leigh syndrome. It indicates that SURF1 mutations might be a common cause of Leigh syndrome in China. The etiology of Leigh syndrome in Chinese patients represents a persistent challenge to clinicians.

[Analysis of ARSA mutations in a Chinese family with metachromatic leukodystrophy].

OBJECTIVE: To identify arylsulftase A gene (ARSA) mutations in a Chinese family with MLD. METHODS: There were two MLD patients in the investigated family. The proband, an 11-year-old girl, was well until the age of 5 years, when she began to experience difficult walking and mental regression. Magnetic resonance imaging (MRI) of her brain showed widespread demyelination, nerve conduction velocity reduced, and ASA activity measured in white blood cells was zero. So, the child was diagnosed having MLD. The proband's young brother also got the same phenotype except clinical symptom being milder than hers. Their parents and elder sister all had normal phenotypes. Genomic DNA samples were extracted from peripheral bloods of the proband and all her family members. All 8 exons and exon-intron boundaries of ARSA gene were amplified by polymerase chain reaction (PCR) and followed by direct DNA sequencing. RESULTS: Two heterozygous mutations of ARSA, which were named as, G251A (R84Q) and G296T (G99V) were identified in the proband. The two mutations were located in exon 2. The same genotype was found in the proband's young brother, but these mutations were not detected in proband's elder sister. The proband's mother had the heterozygous mutations G296T (G99V), and her father had the heterozygous mutation G251A (R84Q). CONCLUSION: These two MLD patients are with both compound heterozygous mutations, which mean one allele with the G296T (G99V) mutation was from their mother, and the other allele with the G251A (R84Q) mutation got from their father. The parents are both carrier with normal phenotype.

The genotype and phenotype studies of 40 Chinese patients with X-linked adrenoleukodystrophy (X-ALD).

OBJECTIVE: To elucidate the phenotype and the genotype-phenotype correlations in Chinese patients with X-linked adrenoleukodystrophy (X-ALD). METHODS: Clinical features of 40 Chinese patients with X-ALD were studied and mutation spectrums were investigated by polymerase chain reaction (PCR) and sequencing. RESULTS: Among these patients, four were siblings from two unrelated families, the others were unrelated. There were 31 cases with childhood cerebral (CCALD), 8 cases with adolescent cerebral (ACALD) and 1 case with adrenomyeloneuropathy (AMN). Visual impairment, which presented in 12 cases (30%), was the most common initial symptom. Nine (69%) of 13 cases who had hydrocortisone and ACTH measured showed adrenal insufficiency. By follow-up date, 19 cases (47.5%) were dead. The interval from onset to death varied from 1 to 6 years and the average were 3.3 years. The mean age at death was 10.5 years. Eleven cases (27.5%) were in vegetable state. The mean interval from onset to apparently vegetable state was 2.8 years (range from 1 to 6 years). Four cases had progressive neurological disability. Four cases were lost follow-up. One case with CCALD and one case with ACALD progressed slowly. The courses of the disease of these two patients were 5 years and 15 years respectively. Thirty five mutations were identified in 40 cases. Most were located within exon 1-3 (40%, 16/40) and exon 6-8 (42%, 17/40). There is a distinct clustering of missense mutations in exon 6 (17%, 7/40). Five types of mutations were associated with CCALD, three with ACALD and a missense mutation was identified in the patients with AMN. The two patients with long disease courses had a missense mutation c.1559 T>A and a nonsense mutation c.1785 G>A respectively. The siblings with similar manifestations and onset age were observed in two families, whose mutations were c.887 A>G and c.1028 G>T. CONCLUSION: The phenotypes, disease severity and rate of neurodegeneration could not be predicted by the nature of mutations.

[Knocking down rat Mecp2 expression by RNAi].

OBJECTIVE: To find the valid siRNA (small interference RNA) sequence to knock down rat Mecp2 expression for the analysis of Mecp2 function by RNA interference (RNAi). METHODS: A plasmid (pMecp2-RFP) expressing rat Mecp2 and marker gene red fluorescent protein (RFP) as a fusion gene was constracted. We first selected a candidate valid sequence by cotransfecting the pMecp2-RFP with 4 candidate siRNAs targeting rat Mecp2 to HEK293T cells respectively, and then identified the RNAi efficiency of the candidate valid siRNA by detecting its effect on suppressing the expression of endogenous rat Mecp2 in PC12 cells. RESULTS: siRNA sequence (5'-GCUGUGAAGGAAUCUUCUA-3') targeting rat Mecp2 mRNA 918-936nt was the valid sequence to knock down rat Mecp2 expression by RNA interference. As the target sequence was located in exon 4 of rat Mecp2 mRNA, we assumed that it could suppress the expression of Mecp2alpha and Mecp2beta at the same time. And as the target sequence was located in the coding region of rat Mecp2, we assumed that it could suppress the expression of both 1.9 kb and 10 kb of rat Mecp2 transcript. CONCLUSION: This study has laid the foundation for constructing rat Mecp2 knocked-down neuronal cell model to study the gene function of Mecp2 in brain development.