Rett综合征X染色体失活方式及失活X染色体起源研究

目的研究Rett综合征（RTT）患儿和其母亲X染色体失活（XCI）方式,患儿失活X染色体来源,探讨RTT XCI与基因型、表型和遗传方式之间的关系。方法对55例RTT患儿、53例RTT患儿的母亲、48例正常女性对照,提取其周围血白细胞DNA,经甲基化敏感性限制性核酸内切酶HpaⅡ消化,对消化前后雄激素受体（AR）基因片段行PCR扩增和基因扫描分析。结果RTT患儿、RTT患儿母亲、正常对照AR基因片段杂合率分别为82％、77％、83％。RTT患儿XCI分布方式与患儿母亲及对照组相比,差异有统计学意义（P〈0．05）,RTT患儿母亲与正常对照组相比,差异无统计学意义（P〉0．05）。与患儿母亲及对照组相比,RTT患儿在50：50～59：41区段内患儿人数显著减少,差异有统计学意义（P〈0．05）。RTT患儿非随机失活在XCI≥65：35和≥80：20范围与母亲及对照组相比人数增多,但差异无统计学意义（P〉0．05）。在21例非随机失活的患儿中,18例倾向于父源X染色体失活,占85．7％,3例倾向于母源。在同一突变位点可以观察到各种XCI方式,T158M高度非随机失活率略高,R133C无高度非随机失活。非典型RTT比典型RTT高度非随机失活率增高,其中保留语言型与先天型相比,高度非随机失活率略高。结论RTT患儿XCI分布方式与及母亲及对照组相比,完全随机失活人数显著减少,但在非随机失活人数上差异无统计学意义。母亲作为携带者传递致病基因不是RTT主要的遗传方式。RTT患儿非随机失活的X染色体主要起源于父亲。RTT患儿的XCI方式与临床表型有一定相关性,但XCI并不能完全解释表型。

X chromosome inactivation patterns in patients with Rett syndrome and their mothers and the parental origin of the priority inactive X chromosome

OBJECTIVE: Rett 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. METHODS: Genomic 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. RESULTS: The 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. CONCLUSION: The 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.