Leipprandt J R, Kraemer S A, Haithcock B E, Chen H, Dyme J L, Cavanagh K T, Friderici K H, Jones M Z
Department of Pathology, Michigan State University, East Lansing 48824, USA.
Genomics. 1996 Oct 1;37(1):51-6. doi: 10.1006/geno.1996.0519.
The complete sequence of the caprine beta-mannosidase cDNA coding region has been determined, and a mutation that is associated with caprine beta-mannosidosis has been identified. Reverse transcriptase-polymerase chain reactions were performed using primers based on bovine and, later, goat cDNA sequences to produce an overlapping series of amplicons covering the entire coding region. The composite cDNA codes for an 879-amino-acid peptide that has four potential N-glycosylation sites. Comparison of the caprine and bovine cDNAs reveals that 96.3% of the nucleotides and 95.2% of the deduced amino acids are identical. A single-base deletion at position 1398 of the coding sequence was identified in the cDNA isolated from a goat affected with beta-mannosidosis. This deletion results in a shift in the reading frame and a premature termination of translation, yielding a deduced peptide of 481 amino acids. An assay, developed to determine the presence or absence of this mutation, confirmed that animals affected with beta-mannosidosis were homozygous for the mutation and that obligate carriers in a caprine beta-mannosidosis colony were heterozygous. This assay accurately distinguished between mutation carrier and noncarrier goats and was used for prenatal diagnosis using DNA collected from fetal fluids. The assay also confirmed chimerism in a goat with an atypically mild beta-mannosidosis phenotype. Thus, this application enables assessment of the efficacy of engraftment of hematopoietic stem cells after prenatal transfer from donor sources.
已确定山羊β-甘露糖苷酶cDNA编码区的完整序列，并鉴定出一种与山羊β-甘露糖苷贮积症相关的突变。使用基于牛和后来山羊cDNA序列的引物进行逆转录聚合酶链反应，以产生覆盖整个编码区的重叠扩增子系列。复合cDNA编码一个具有四个潜在N-糖基化位点的879个氨基酸的肽。山羊和牛cDNA的比较显示，96.3%的核苷酸和95.2%的推导氨基酸是相同的。在从一只患有β-甘露糖苷贮积症的山羊分离的cDNA中，在编码序列的第1398位鉴定出一个单碱基缺失。这种缺失导致阅读框移位和翻译提前终止，产生一个推导的481个氨基酸的肽。为确定该突变的存在与否而开发的一种检测方法证实，患有β-甘露糖苷贮积症的动物对该突变是纯合的，并且在一个山羊β-甘露糖苷贮积症群体中必然携带者是杂合的。该检测方法准确地区分了突变携带者和非携带者山羊，并用于使用从羊水收集的DNA进行产前诊断。该检测方法还证实了一只具有非典型轻度β-甘露糖苷贮积症表型的山羊存在嵌合体。因此，这种应用能够评估产前从供体来源转移造血干细胞后的植入效果。
