Wu Xr

Large deletions of the MECP2 gene in Chinese patients with classical Rett syndrome

To the Editor: Rett syndrome (RTT; MIM 312750), an Xlinked disorder that almost exclusively affects girls (1), is caused by mutations in the MECP2 (methyl CpG binding protein 2) gene (2). DNA sequencing identifies mutations in the MECP2 gene in 80% of classic RTT patients. Recently, quantitative analysis has identified large deletions within theMECP2 gene in 20–38% of those RTT patients with no mutation found on sequencing (3–9). We have studied 30 Chinese classical RTT patients without a defined MECP2 mutation using multiplex ligase-dependent probe amplification (MLPA) to detect large deletions of the MECP2 gene. These 30 patients were referred from 11 provinces of China. All patients fulfilled the international diagnostic criteria (10) and did not have a defined MECP2 mutation. Genomic DNA was prepared and purified from peripheral blood (Qiagen, Valencia, CA). Informed consent was obtained. MECP2-MLPA (covering each of the four exons of the MECP2 gene) was performed using kit P015C (MRC-Holland,Amsterdam) (11). X chromosome inactivation (XCI) was tested in all 11patientswith a large deletion in theMECP2 gene by analysing the androgen receptor gene (AR) in peripheral blood DNA (12). X inactivation was considered significantly skewed if the ratio equated or exceeded 80:20. We detected 11 cases with large deletions of the MECP2 gene in the 30 Chinese classical RTT patients (36.6%). Ten of the 11 deletions involved either exon 3 or both exons 3 and 4 (Table 1). In one case (R-111), the flanking IRAK1 gene was deleted along with the exons 3 and 4. The clinical features, the MLPA and XCI results of these 11 patients are summarized in Table 1. This is the first report on the study of large deletions of the MECP2 gene in Chinese patients with classical RTT. A review of the literature showed nine studies on large deletions of the MECP2 gene on Caucasian classic RTT patients with a detection rate of 20–38% in those with no mutation found on sequencing (3–9, 13). Little is known about Chinese patients with RTT. We reported previously the identification of 17 cases with aMECP2mutation among 30 Chinese RTT patients by DNA sequencing (14). The present study identified 11 cases with large deletion in 30 Chinese classical RTT patients without MECP2 gene mutation on sequencing, a detection rate (36.6% or 11/30) comparable to the reported data. In other studies, large deletions frequently involve either exon 4 or both exons 3 and 4 of the MECP2 gene (4, 5, 7–9). Our experience is consistent with previous observations. Archer et al. (8) reported five RTT patients with additional congenital anomalies, accounting for 22.7% of those with large deletions involving the downstream DNA sequences. Deletions involving the adjacent IRAK1 gene and other genes were proposed as the cause of congenital anomalies. Ravn et al. (7) reported larger deletions involving the downstream IRAK1 gene in three patients, who did not display additional congenital anomalies or other clinical features. Our patient (R-111) with a deletion involving exons 3 and 4 ofMECP2 gene as well as the IRAK1 gene does not have congenital anomalies or other clinical features. Based on these data, the cause of the congenital anomalies may not be an IRAK1 gene deletion. Of our 11 patients aged between 3 and 23 years, the severity scores according to Kerr and Archer (8, 15), vary from4 to 8. Because some of the symptoms are age-dependent and the number of subjects in this study is small, we were unable to establish any correlation between phenotype and different exon deletions of the MECP2 gene. In addition, skewed XCI pattern was found in two RTT patients (Table 1). Because of the small number of patients with large deletions in our study, we did not attempt to correlate the degree of XIC with the type/size of the MECP2 deletion. Our experience is that MLPA, as a complement to DNA sequencing, is a useful tool for Rett syndrome molecular diagnosis especially in countries with a big population like China, where the number of patients requiring analysis can be large.

Genotype/phenotype analysis in Chinese laminin‐α2 deficient congenital muscular dystrophy patients

Laminin‐α2 deficient congenital muscular dystrophy (CMD) is an autosomal recessive disorder characterized by severe muscular dystrophy, which is typically associated with abnormal white matter. In this study, we assessed 43 CMD patients with typical white matter abnormality and laminin‐α2 deficiency (complete or partial) diagnosed by immunohistochemistry to determine the clinical and molecular genetic characteristics of laminin‐α2 deficient CMD. LAMA2 gene mutation analysis was performed by direct sequencing of genomic DNAs. Exonic deletion or duplication was identified by multiplex ligation‐dependent probe amplification (MLPA) and verified by high‐density oligonucleotide‐based CGH microarrays. Gene mutation analysis revealed 86 LAMA2 mutations (100%); 15 known and 37 novel. Among these mutations, 73.9% were nonsense, splice‐site or frameshift and 18.8% were deletions of one or more exons. Genetic characterization of affected families will be valuable in prenatal diagnosis of CMD in the Chinese population.

RNAi-induced down-regulation of Mecp2 expression in the rat brain

The MECP2 (methyl‐CpG‐binding protein 2) gene has been implicated in the pathogenesis of Rett Syndrome. A rat model with a down‐regulated Mecp2 was established using a recombinant lentiviral vector expressing small hairpin RNA of the rat Mecp2 gene. Four recombinant vectors were constructed by inserting sequences of small hairpin RNA targeting the rat Mecp2 gene. To determine which vector resulted in optimal down‐regulation, rat skin fibroblasts were transfected with four recombinant vectors, respectively. The recombinant vector mecp2‐sh‐1 decreased expression of Mecp2 mRNA and protein relative to the Control group. 12–24 h after birth, neonatal rat pups were divided into three groups, Re‐Lenti group (injected with recombinant lentiviral vector, mecp2‐sh‐1), Lenti group (injected with lentivirus containing no inserted sequences) and a non‐injected Control group (Control group). Rats in the injection groups were given intraventricular injections. 7 days and 21 days following injection, no inflammation was observed in the rat brain in the two injected groups, whereas EGFP expression was readily detectable. Sensory‐motor reflexes were transiently abnormal in Re‐Lenti group, whereas no abnormality was observed in either Control group. Mecp2 mRNA was lower in the hippocampus and cerebral cortex in the Re‐Lenti group relative to the Lenti and Control groups. Although no typical RTT like symptoms were observed, the neonatal rats injected with recombinant lentivirus displayed some transient neurobehavioral abnormalities during early development. Bdnf mRNA expression decreased in the hippocampus, supporting the hypothesis that Bdnf may be a target gene of MeCP2 in the CNS.

Gene delivery into primary cerebral cortical neurons by lentiviral vector

Several studies have shown the ability of human immunodeficiency virus type 1 (HIV‐1)‐based lentiviral vectors to infect nondividing brain neurons. We are the first to show that primary embryonic cerebral cortical neurons can be efficiently transduced by an HIV‐1‐based lentiviral vector encoding enhanced green fluorescent protein (EGFP). We also describe the optimal conditions for the transduction of cerebral cortical neurons with lentiviral vectors, and the kinetic process of infection. The percentage of cells expressing EGFP is a function of the time in culture and virus dose. The highest percentage of EGFP‐expression achieved was 46.77% at 4 days in vitro (DIV) with a multiplicity of infection (m.o.i.) of 20. The results show that lentiviral vectors are not only good prospects for in vivo gene delivery, but are also good candidates for in vitro studies of the function of gene products in primary cerebral cortical neurons.