Li Mr

MECP2 and CDKL5 gene mutation analysis in Chinese patients with Rett syndrome

AbstractRett syndrome (RTT) is a progressive neurodevelopmental disorder that is caused by mutations in the X-linked methyl-CpG-binding protein2 (MECP2) gene. In this study, the MECP2 sequences in 121 unrelated Chinese patients with classical or atypical RTT were screened for deletions and mutations. In all, we identified 45 different MECP2 mutations in 102 of these RTT patients. The p. T158M mutation (15.7%) was the most common, followed in order of frequency by p. R168X (11.8%), p. R133C (6.9%), p. R270X (6.9%), p. G269fs (6.9%), p. R255X (4.9%), and p. R306C (3.9%). In addition, we identified five novel MECP2 mutations: three missense (p. K305E, p. V122M, p. A358T), one insertion (c.45-46insGGAGGA), and one 22 bp deletion (c.881-902del22). Large deletions represented 10.5% of all identified MECP2 mutations. Conversely, mutations in exon 1 appeared to be rare (0.9%). The remaining cases without MECP2 mutations were screened for the cyclin-dependent kinase-like 5 (CDKL5) gene using denaturing high-performance liquid chromatography (DHPLC). One synonymous mutation (p. I72I) was found in exon 5, suggesting that CDKL5 is a rare cause of RTT. The overall MECP2 mutation detection rate for this patient series was 84.3:87.9% in 107 classical RTT cases and 57.1% in 14 atypical RTT cases. Moreover, there were two patients with homozygous mutations and normal female karyotypes. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases.

X chromosome inactivation in Rett Syndrome and its correlations with MECP2 mutations and phenotype.

Rett syndrome (RTT) is an X-linked dominant neurodevelopment disorder, which is mainly caused by gene mutation of methyl-CpG-binding protein 2 (MECP2). The correlations between genotype, X chromosome inactivation (XCI), and phenotype have been studied, but the results are conflicting. In the present study, XCI patterns in patients and their mothers, parental origin of skewed X chromosome in patients, and the correlations between XCI, genotype, and phenotype were analyzed in 52 cases of RTT with MECP2 mutations, 50 RTT mothers, and 48 normal female controls. The results showed XCI and genotype had limitations in explaining all the phenotypic manifestations of RTT. Other genomic factors have to be considered to explain the phenotypic differences.

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.

Analysis of the Parental Origin of De Novo MECP2 Mutations and X Chromosome Inactivation in 24 Sporadic Patients With Rett Syndrome in China

Rett syndrome is an X-linked neurodevelopmental disorder that predominantly affects females. It is caused by mutations in methyl-CpG-binding protein 2 gene. Due to the sex-limited expression, it has been suggested that de novo X-linked mutations may exclusively occur in male germ cells and thus only females are affected. In this study, the authors have analyzed the parental origin of mutations and the X-chromosome inactivation status in 24 sporadic patients with identified methyl-CpG-binding protein2 gene mutations. The results showed that 22 of 24 patients have a paternal origin. Only 2 patients have a maternal origin. Except for 2 cases which were homozygotic at the androgen receptor gene locus, of the remaining 22 cases, 16 cases have a random X-chromosome inactivation pattern; the other 6 cases have a skewed X-chromosome inactivation and they favor expression of the wild allele. The relationship between X-chromosome inactivation and phenotype may need more cases to explore.

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.

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.