In vitro expression study of novel mutations in phenylalanine hydroxylase gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the in vitro expression of 6 novel missense mutations (R270G, P275A, F121L, A156P, E183G, I324N) and a previously described R408Q mutation of phenylalanine hydroxylase (PAH) gene and explore the genotype-phenotype correlation through comparison of protein levels and residual enzyme activities.</p><p><b>METHODS</b>Seven expression vectors containing PAH cDNA were constructed with a site-directed mutagenesis kit. The plasmids were extracted and sequenced to confirm the target mutations. pcDNA3.0 containing PAH cDNA was transfected into COS-7 cells and total proteins were extracted 48 h after transfection. The quantities of proteins and residual enzyme activities of the 7 mutants were assessed with the wild-type PAH gene as reference.</p><p><b>RESULTS</b>Relative quantities of PAH proteins for R270G, P275A, F121L, A156P, E183G, I324N and R408Q were 10.5%, 56.6%, 54.3%, 8.7%, 8.5%, 67.3% and 85.4%, respectively. The residual enzyme activities were 7.7%, 27.6%, 19.0%, 10.4%, 9.1%, 50.6% and 40.2%, respectively.</p><p><b>CONCLUSION</b>PAH residual enzyme activities of 7 PAH mutants were all significantly reduced.</p>

Prenatal diagnosis of mucopolysaccharidosis type II / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method of iduronate-2-sulfatase (IDS) activity assay and mutation analysis of IDS gene for the prenatal diagnosis of mucopolysaccharidosis type II (MPSII).</p><p><b>METHODS</b>Prenatal diagnosis of two cases was performed using cultured fetal amniotic fluid cells. Enzyme activity of IDS in cultured fetal amniotic fluid cells extracted from the two pregnant women at high risk of MPS II was measured. Meanwhile, genomic DNA was extracted for fetal gender testing and mutation analysis of the IDS gene.</p><p><b>RESULTS</b>Enzyme activity assay showed that IDS activity in amniotic fluid cells was significantly decreased. IDS gene sequencing showed that the male fetus was hemizygous mutant, and the female fetus was carrier of heterozygous mutation. Therefore the male fetus was an MPS II patient and the female fetus was a mutation carrier.</p><p><b>CONCLUSION</b>Determination of IDS activity in fetal amniotic fluid cells together with IDS gene mutation analysis is a rapid, sensitive and accurate method of prenatal diagnosis of MPS II. Using this method, prenatal diagnosis for pregnant women at high risk of MPSII can be achieved.</p>