Ben-Yoseph Y, Mitchell D A
Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan.
Am J Med Sci. 1995 Feb;309(2):88-91. doi: 10.1097/00000441-199502000-00006.
Mutations causing metachromatic leukodystrophy and pseudo-deficiency were detected in the arylsulfatase A gene by methods based on different wild-type and mutant restriction sites. After polymerase chain reaction amplification of fragments of the arylsulfatase A gene and digestion by the appropriate endonuclease, the mixtures were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The common splice mutation in intron 2 (459 + 1G-->A) causing, in homozygosity, late-infantile metachromatic leukodystrophy and the common missense mutation in exon 8 (P426L) causing, in homozygosity, adult or juvenile metachromatic leukodystrophy were found to abolish Bst NI and Aci I sites, respectively. The polyadenylation pseudo-deficiency mutation (1619A-->G) was found to create a Mae III restriction site. The N-glycosylation pseudo-deficiency mutation (N350S) does not produce or destroy any known restriction site, and in this case, introduction of a single nucleotide mismatch in one of the primers enabled the authors to create a Bfa I site in the mutant allele.
通过基于不同野生型和突变型限制性位点的方法，在芳基硫酸酯酶A基因中检测到了导致异染性脑白质营养不良和假缺陷的突变。在对芳基硫酸酯酶A基因片段进行聚合酶链反应扩增并经适当的核酸内切酶消化后，混合物通过聚丙烯酰胺凝胶电泳进行分离，并用溴化乙锭染色进行可视化。发现内含子2中的常见剪接突变（459 + 1G→A），纯合时导致晚发性婴儿异染性脑白质营养不良，以及外显子8中的常见错义突变（P426L），纯合时导致成人或青少年异染性脑白质营养不良，分别消除了Bst NI和Aci I位点。发现聚腺苷酸化假缺陷突变（1619A→G）产生了一个Mae III限制性位点。N-糖基化假缺陷突变（N350S）不会产生或破坏任何已知的限制性位点，在这种情况下，在其中一个引物中引入单个核苷酸错配使作者能够在突变等位基因中创建一个Bfa I位点。
