Tomatsu S, Fukuda S, Cooper A, Wraith J E, Uchiyama A, Hori T, Nakashima Y, Yamada N, Sukegawa K, Kondo N
Department of Pediatrics, Gifu University School of Medicine, Japan.
Hum Genet. 1995 Apr;95(4):376-81. doi: 10.1007/BF00208958.
Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetyl-galactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the full-length cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5'-end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.
利用Southern印迹分析法，对48例IVA型黏多糖贮积症患者的96个等位基因（36个日本患者来源的等位基因和60个高加索人来源的等位基因）进行了结构基因改变的研究。所有患者先前均已证实缺乏N-乙酰半乳糖胺-6-硫酸酯硫酸酯酶，且临床表现出广泛的严重程度差异。最初，以全长cDNA为探针，36条来自日本患者的染色体中有5条在用BamHI、SacI和XhoI消化的DNA方面显示出相似的重排。随后使用覆盖整个基因的7个基因组片段进行分析，增强了对上述限制酶产生的异常片段的检测。相反，用这些方法分析时，60条高加索人来源的染色体未显示出大的结构重排迹象。这两个人群之间存在统计学上的显著差异（P < 0.01）。一名病情严重的日本患者通过BamHI印迹法在两条染色体上均显示出结构重排。正常个体中存在的一个８.０kb片段和一个高度多态性的7.0kb至11.0kb片段消失，观察到两个异常片段，大小分别为11.5kb和12.0kb。另外三名日本患者除了正常片段模式外也显示出这两个异常片段，因此在这种重排方面是杂合子。由于串联重复元件数量可变导致多态性条带复杂，Southern印迹的解读很困难。然而，通过利用这些异常片段或多态性条带，即使在突变特征不明确的家族中，携带者检测也是有效的。用探针MG-A（内含子1中的5'-端基因组探针）杂交显示，一名日本患者和一名高加索患者的BamHI印迹中有一个8.4kb片段；XhoI、SacI和EcoRI印迹正常。由于在一名正常对照中也观察到了这种BamHI改变，它似乎是一种罕见的非病理性多态性。
