Clemens P R, Ward P A, Caskey C T, Bulman D E, Fenwick R G
Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030.
Neurology. 1992 Sep;42(9):1775-82. doi: 10.1212/wnl.42.9.1775.
We identified a premature chain termination mutation in two brothers with Duchenne muscular dystrophy and correlated the mutation in one of the brothers with immunologic detection of dystrophin in skeletal muscle. Southern and polymerase chain reaction (PCR) studies of genomic DNA from the affected boys showed no major gene rearrangements. However, the noted absence of a HindIII Southern fragment containing the proximal portion of exon 48 led to the identification of a point mutation that creates a new HindIII restriction site in that exon. Exon 48 was amplified by PCR from DNA of the patients and other family members and digested with HindIII to show the mutation in the two boys and also in their mother and maternal grandmother. Direct DNA sequencing demonstrated a cytosine-to-thymine transition at nucleotide 7163 of dystrophin that converts a glutamine codon (CAA) to an ochre chain termination codon (UAA). This mutation predicts a truncated dystrophin missing the distal spectrin-like repeat region, the cysteine-rich domain, and the carboxy terminal. Immunohistochemistry of skeletal muscle from one of the affected boys revealed membrane-localized dystrophin in the majority of fibers detected by anti-dystrophin antibodies against (1) the amino terminal and (2) part of the spectrin-like repeat region; both regions would be present in the truncated dystrophin predicted by the chain termination mutation. This suggests that the carboxy terminal may not be an absolute requirement for dystrophin membrane localization. Very few muscle fibers also showed peripheral immunostaining using anti-dystrophin antibodies against the carboxy terminal, suggesting gene reversion, suppression, or read-through in these rare fibers.
我们在两名患有杜氏肌营养不良症的兄弟中发现了一个提前链终止突变，并将其中一名兄弟的突变与骨骼肌中抗肌萎缩蛋白的免疫学检测相关联。对患病男孩的基因组DNA进行的Southern杂交和聚合酶链反应（PCR）研究未发现主要的基因重排。然而，注意到含有外显子48近端部分的HindIII Southern片段缺失，这导致鉴定出一个点突变，该突变在该外显子中产生了一个新的HindIII限制性位点。通过PCR从患者和其他家庭成员的DNA中扩增出外显子48，并用HindIII消化，以显示两名男孩及其母亲和外祖母中的突变。直接DNA测序显示，抗肌萎缩蛋白第7163位核苷酸处发生了胞嘧啶到胸腺嘧啶的转变，将谷氨酰胺密码子（CAA）转换为赭石链终止密码子（UAA）。该突变预测会产生一个截短的抗肌萎缩蛋白，缺失远端血影蛋白样重复区域、富含半胱氨酸的结构域和羧基末端。对其中一名患病男孩的骨骼肌进行免疫组织化学分析，发现抗肌萎缩蛋白抗体检测到的大多数纤维中，抗肌萎缩蛋白定位于细胞膜，这些抗体分别针对（1）氨基末端和（2）部分血影蛋白样重复区域；这两个区域都将存在于链终止突变预测的截短抗肌萎缩蛋白中。这表明羧基末端可能不是抗肌萎缩蛋白定位于细胞膜的绝对必要条件。极少数肌纤维使用针对羧基末端的抗肌萎缩蛋白抗体也显示出外周免疫染色，提示这些罕见纤维中存在基因回复、抑制或通读现象。
