OBJECTIVERett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder that affects females almost exclusively, caused by mutations in MECP2 gene on chromosome Xq28, with symptoms such as autism, severe mental deficiency, deceleration of head growth, ataxia, loss of purposeful hand function and characteristic stereotypic hand movements. Over 80% MECP2 mutations located in the exon 3 and exon 4 were confirmed by our work and large-scale studies. RTT is defined based on clinical presentation. It is difficult to diagnose in the early life without definite biochemical abnormality, but genetic test is helpful for this. The aim of this study was to investigate the feasibility and clinical significance of applying long range polymerase chain reaction (PCR) to RTT diagnosis and establish a simple, economic, efficient method of genetic diagnosis.

METHODGenomic DNA was extracted using standard procedures from the peripheral blood leukocytes of each patient. Long range polymerase chain reaction(PCR)and DNA direct sequencing were employed to analyze the exon 3 and 4 of MECP2 gene simultaneity in 40 patients with RTT. The PCR products were checked by using 1.5% agarose gel.

RESULTIn total, 18 different MECP2 mutations were identified in 33 of the 40 diagnosed sporadic female patients with RTT. Missense mutations were 16, followed by 14 nonsense mutations and 3 deletions. The 314 base pairs large deletion was identified. The p. T158M mutation (21%, 7/33) was the most common, followed in order of frequency by p. R255X (12%, 4/33), p. R168X and p. R106W (9%, 3/33) respectively, p. R270X and p. Y141X (6%, 2/33) respectively, p. R133C, p. D156H, p. P157L, p. P225R, p. Q244X, p. Q262X, p. R294X, p. R306C, P322L, c. 1005del G, c.1005-1318del 314 bp and c.1127-1179del 53 bp (3%, 1/33), respectively.

CONCLUSIONLong range PCR is a simple, economic, quick, precise method of genetic diagnosis and was able to find 83% MECP2 gene mutations in RTT patients in this study. It is helpful for RTT clinical diagnosis in early stage. On the other hand, it may detect recurrent mutations and large deletions at the same time.

Child ; Child, Preschool ; DNA ; analysis ; Exons ; genetics ; Female ; Humans ; Methyl-CpG-Binding Protein 2 ; genetics ; Mutation ; Polymerase Chain Reaction ; methods ; Rett Syndrome ; diagnosis ; genetics

OBJECTIVERett syndrome (RTT) is a severe childhood neurodevelopmental disorder mainly affecting females. The pathogenic gene is located at Xq28, which codes for the methyl-CpG-binding protein 2. MECP2 gene is affected by X chromosome inactivation (XCI). The different XCI patterns of females could affect the expression ratios of pathogenic gene, causing changes in clinical symptoms. In order to understand the XCI patterns in RTT patients and the relationship between XCI pattern, genotype and phenotype, the XCI patterns in patients with RTT and their mothers, the parental origin of the priority inactive X chromosome in RTT, and the relations of XCI patterns with genotype and phenotype in RTT cases were analyzed.

METHODSGenomic DNA was extracted from peripheral blood of 55 cases with RTT (52 with MECP2 mutations, 3 without mutations), 53 mothers of RTT cases and 48 normal female controls. DNA was digested with methylation sensitive restriction endonuclease Hpa II. Then the undigested and digested DNAs were amplified via PCR for the first exon of human androgen receptor (AR) gene. PCR products were analyzed by Genescan.

RESULTSThe heterozygotic rates of AR gene were 82%, 77% and 83% in RTT patients, mothers and controls, respectively. XCI distribution pattern of RTT was different from that of the mothers and control, P < 0.05. More mothers and controls than RTT patients were in the area of XCI 50:50 - 59:41. The differences between them were statistically significant (P < 0.05). No significant difference in XCI distribution patterns between mothers and the control groups was found (P > 0.05). Non-random XCI rates in the areas of XCI >or= 65:35 and >or= 80:20 were 53.35% and 17.8%, respectively, in RTT patients, compared with the mothers group (36.6%, 7.3%) and control group (35%, 10%), it was higher in RTT patients, but the difference was not statistically significant (P > 0.05). In 18 of 21 cases with XCI >or= 65:35, the priority inactive X chromosome was of paternal origin (85.7%). Variable XCI patterns were observed in the same gene mutation patients. The highly skewed XCI as well as the random XCI were found in patients with mild, severe and typical phenotype. The rate of highly skewed XCI in atypical patients was higher than that in typical RTT patients. The rate of highly skewed XCI in T158M was higher than the other type mutations. No highly skewed XCI was observed in cases with R133C mutation.

CONCLUSIONThe XCI distribution pattern of RTT patients was different from that of RTT mother and control groups. There was no significant difference in XCI distribution patterns between mothers and the control groups. It was not a main genetic pattern in RTT that mothers as the carriers to transmit the pathogenic gene to the patients. Non-random XCI was not the main XCI pattern in RTT patients. The priority inactive X chromosome was mainly of paternal origin. XCI could modify the clinical phenotype of RTT, but had limitations in explaining all the phenotypes manifested in RTT cases.

Adolescent ; Adult ; Child ; Child, Preschool ; Chromosomes, Human, X ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Heterozygote ; Humans ; Mothers ; Mutation ; Phenotype ; Receptors, Androgen ; genetics ; Rett Syndrome ; diagnosis ; genetics ; X Chromosome Inactivation ; genetics

OBJECTIVETo analyze the genetic characteristics and molecular mechanism of Chinese patients with Rett syndrome (RTT) and assess the recurrent risk in order to provide genetic counseling for the family with RTT patient.

METHODSMethyl-CpG-binding protein 2 (MECP2) gene mutation analysis were performed on 405 Chinese RTT cases and 292 mothers of the patients with MECP2 mutations with polymerase chain reaction (PCR), direct sequencing and multiplex ligation-dependent probe amplification (MLPA). Then cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1) genes mutation analysis were performed on the patients without MECP2 mutation. Parental origin of mutated MECP2 gene was detected with allele specific PCR analysis. Based on the difference methylation in CpG island of the first exon of human androgen-receptor gene on active and inactive X-chromosomes, methylation sensitive restriction endonuclease digestion was used to analyze the X-chromosome inactive (XCI) patterns.

RESULTSMECP2 gene mutation was found in 86.9% RTT cases. CDKL5 gene mutation was found in only 3 cases with early-onset seizures variant. No FOXG1 mutation was found. There were 94.4% MECP2 mutations of paternal origin,and point mutations were common. However, microdeletions were common in maternal origin mutation. MECP2 gene mutation was found in only 1 (0.34%,1/292) mother with normal phenotype and non-random XCI pattern. Her daughter was a RTT patient with preserved speech variant, and her XCI pattern was random.

CONCLUSIONMECP2 is the main pathogenic gene in RTT. CDKL5 gene should be screened in patients with early-onset seizures variant without MECP2 gene mutation. The majority of RTT patients had paternally derived de novo MECP2 gene mutation, which may explain the high female to male ratio in RTT. Only 0.34% mothers carried the pathogenic mutation, indicating a lower recurrent risk for RTT families. The XCI may modulate the phenotype of RTT, so MECP2 gene mutation screening in the mothers is important for genetic counseling.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; Child ; Child, Preschool ; Female ; Genetic Counseling ; methods ; Genetic Predisposition to Disease ; Humans ; Methyl-CpG-Binding Protein 2 ; genetics ; Prenatal Diagnosis ; methods ; Rett Syndrome ; genetics ; Young Adult

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1 per 10,000- 15,000 female births worldwide. The disease-causing gene has been identified as MECP2 (methyl- CpG-binding protein 2). In this study, we performed diagnostic mutational analysis of the MECP2 gene in RTT patients. Four exons and a putative promoter of the MECP2 gene were analyzed from the peripheral blood of 43 Korean patients with Rett syndrome by PCR-RFLP and direct sequencing. Mutations were detected in the MECP2 gene in approximately 60.5% of patients (26 cases/43 cases). The mutations consisted of 14 different types, including 9 missense mutations, 4 nonsense mutations and 1 frameshift mutation. Of these, three mutations (G161E, T311M, p385fsX409) were newly identified and were determined to be disease-causing mutations by PCR- RFLP and direct sequencing analysis. Most of the mutations were located within MBD (42.3%) and TRD (50%). T158M, R270X, and R306C mutations were identified at a high frequency. Additionally, an intronic SNP (IVS3+23C>G) was newly identified in three of the patients. IVS3+23C>G may be a disease-related and Korea-specific SNP for RTT. L100V and A201V are apparently disease-causing mutations in Korean RTT, contrary to previous studies. Disease-causing mutations and polymorphisms are important tools for diagnosing RTT in Koreans. The experimental procedures used in this study should be considered for clinical molecular biologic diagnosis.
Rett Syndrome/diagnosis/*genetics ; Polymorphism, Single Nucleotide ; Polymorphism, Restriction Fragment Length ; Polymerase Chain Reaction ; *Mutation ; Molecular Sequence Data ; Methyl-CpG-Binding Protein 2/*genetics ; Male ; Korea ; Humans ; Female ; DNA Mutational Analysis ; Base Sequence