A deletion/insertion mutation in the IDUA gene in a Libyan Jewish patient with Hurler syndrome (mucopolysaeeharidosis IH)
A deletion/insertion mutation in the IDUA gene in a Libyan Jewish patient with Hurler syndrome (mucopolysaeeharidosis IH)
复制标题
患有 Hurler 综合征（粘多糖沉积症 IH）的利比亚犹太患者 IDUA 基因缺失/插入突变
作者：
Samuel M. Moskowitz;P. Tieu;Elizabeth F. Neufeld
粘多糖样变性I（MPS I）包括一组罕见的常染色体隐性疾病，由溶酶体酶（L艾杜糖醛酸酶）缺乏引起，并导致降级的硫酸皮肤素和硫酸乙酰肝素蓄积（Neufeld和Muenzer，1989;霍普伍德和Morris，1990）。MPS 1的临床表型范围从Hurler综合征中的非常严重（MPS IH）到Scheie综合征中的相对良性（MPS IS），中间表型称为HurlerBcheie（MPS IH/S）。通过最近对编码α-艾杜糖醛酸酶的互补和基因组DNA的分离和表征，已经使得分析这些病症背后的突变成为可能（Scott等人，1991，1992 a; Moskowitz等人，1992年a）。已经发现Trp，pTer等位基因在64名MPS I患者中是常见的，其中大多数是盎格鲁-撒克逊血统（Scott et al.，1992年b）。在以色列阿拉伯人（德鲁兹人和穆斯林）社区的Hurler患者中发现了错义突变（Thr，，，+Pro）和两种无义突变（Tyr，，，+Ter和Gln，，，-+Ter）（Moskowitz等人，1992年b）。在MPS I的犬模型中已经发现内含子1供体剪接位点中的G+A转换（Menon等人，1992年）。所有这些突变都是由单碱基变化引起的。我们现在报告一个更复杂的突变，这是在一个利比亚犹太血统的Hurler患者中发现的。将从培养的皮肤成纤维细胞中提取的RNA（Chomczynski和Sacchi，1987）逆转录并扩增（RT-PCR）为六个重叠片段;对于跨越外显子3-7的片段，扩增产生了双联体，其下带略小于505 bp的单个正常带，上带略大于505 bp的单个正常带（图1A）。通过凝胶电泳分离双联体的两条带并进行循环测序（Smith等人，1990年）。下带的序列分析显示外显子6中12个核苷酸的缺失，这是由断裂点处22个核苷酸的缺失和10个核苷酸的插入引起的（未显示）;在PCR扩增的基因组DNA的相应区域中观察到相同的序列（图1B）。从亲本获得的基因组DNA的相同区段的扩增显示出不同大小的两条带以及预期的异源双链体（图1C，上图）。在用HaeIII切割后，更容易看到正常和突变片段之间的大小差异（图1C，下图）;来自亲本的扩增DNA的切割产物显示正常和突变条带，证实了它们对于缺失插入突变的杂合性。父母被认为有相同的突变等位基因，因为他们是第一堂兄弟姐妹。插入似乎是由一个短序列的复制引起的（图1D，下划线）。突变的一个结果是在半胱氨酸205位点产生终止密码子。北方分析（未显示）显示α-L-艾杜糖醛酸酶mRNA水平显著降低，在肌动蛋白mRNA标准化后，通过光密度测定法估计低于正常对照水平的十分之一。该患者中a-L-艾杜糖醛酸酶mRNA的非常低的水平与过早翻译终止通常导致mRNA缺陷的观察结果一致（例如，巴塞加和
Mucopolysaccharidosis I (MPS I) comprises a group of rare autosomal recessive disorders caused by the deficiency of the lysosomal enzyme, a L iduronidase, and the resulting accumulation of undergraded dermatan sulfate and heparan sulfate (Neufeld and Muenzer, 1989; Hopwood and Morris, 1990). The clinical phenotype of MPS 1 ranges from the very severe in Hurler syndrome (MPS IH) to the relatively benign in Scheie syndrome (MPS I S), with an intermediate phenotype designated HurlerBcheie (MPS IH/S). Analysis of mutations underlying these disorders has been made possible by the recent isolation and characterization of complementary and genomic DNAs encoding a ~ iduronidase (Scott et al., 1991, 1992a; Moskowitz e t al., 1992a). A Trp,,pTer allele has been found to be common among 64 patients with MPS I, most of whom were of Anglo-Saxon descent (Scott et al., 1992b). A missense mutation (Thr,,,+Pro) and two nonsense mutations (Tyr,,+Ter and Gln,,,-+Ter) have been found in Hurler patients of the Israeli Arab (Druze and Moslem) communities (Moskowitz et al., 1992b). A G+A transition in the intron 1 donor splice site has been found in the canine model of MPS I (Menon et al., 1992). All of these mutations are caused by single base changes. We now report on a mutation of greater complexity, which has been found in a Hurler patient of Libyan Jewish origin. RNA extracted from cultured skin fibroblasts (Chomczynski and Sacchi, 1987) was reverse transcribed and amplified (RT-PCR) in six overlapping segments; for the segment spanning exons 3-7, the amplification yielded a doublet with the lower band slightly smaller and the upper band slightly larger than the single normal band of 505 bp (Fig. IA). The two bands of the doublet were separated by gel electrophoresis and subjected to cycle sequencing (Smith et al., 1990). Sequence analysis of the lower band showed a loss of 12 nucleotides in exon 6, caused by a deletion of 22 nt and an insertion of 10 nt at the breakpoint (not shown); an identical sequence was seen in the corresponding region of PCR-amplified genomic DNA (Fig. 1B). Amplification of the same segment of genomic DNA obtained from the parents shows two bands of different size as well as the expected heteroduplex (Fig. IC, upper panel). The size difference between normal and mutant segments is more easily seen after cleavage with HaeIII (Fig. IC, lower panel); the cleavage products of the amplified DNA from the parents show both normal and mutant bands, confirming their heterozygosity for the deletiodinsertion mutation. The parents were expected to have the same mutant allele since they are first cousins. The insertion appears to have arisen by duplication of a short sequence (Fig. lD, underlined). One result of the mutation is to create a termination codon at the site of cysteine 205. Northern analysis (not shown) showed a markedly reduced level of a-L-iduronidase mRNA, estimated by densitometry to be less than a tenth the level in the normal control after normalization for actin mRNA. The very low level of a-L-iduronidase mRNA in this patient is consistent with the observation that premature translation termination often leads to mRNA deficiency (e.g., Baserga and