Objective: Rett 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.
Method: Genomic 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.
Result: In 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.
Conclusion: Long 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.