Hirschhorn R, Tzall S, Ellenbogen A
Department of Medicine, New York University Medical School, NY 10016.
Proc Natl Acad Sci U S A. 1990 Aug;87(16):6171-5. doi: 10.1073/pnas.87.16.6171.
We have previously characterized mutant adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) enzymes in seven children with partial ADA deficiency. Six children shared common origins, suggesting a common progenitor. However, we found evidence for multiple phenotypically different mutant enzymes. We hypothesized that many of the mutations would be at CpG dinucleotides, hot spots at which spontaneous deamination of 5-methylcytosine results in C to T or G to A transitions. Digestion of DNA from these children with Msp I and Taq I, enzymes recognizing CpG dinucleotides, identified three different mutations, each correlating with expression of a different mutant enzyme. Sequencing of cDNA clones and genomic DNA amplified by polymerase chain reaction confirmed the presence of C to T or G to A transitions at CpG dinucleotides (C226 to T, G446 to A, and C821 to T, resulting in Arg76 to Trp, Arg149 to Gln, and Pro274 to Leu). A "null" mutation, also found in two ADA-deficient severe combined immunodeficient children, was serendipitously detected as gain of a site for Msp I. Simultaneous loss of a site for Bal I defined the precise base substitution (T320 to C, Leu107 to Pro), confirmed by sequence analysis. To determine the true frequency of hot spot mutation in these children, consecutively ascertained through a newborn screening program, we sequenced cDNA from the remaining alleles. Two others were hot spot mutations (C631 to T and G643 to A, resulting in Arg211 to Cys and Ala215 to Thr), each again resulting in expression of a phenotypically different mutant enzyme. Only one additional mutation (previously identified by us) is not in a hot spot. These seven mutations account for all 14 chromosomes in these children. There is thus a very high frequency of hot spot mutations in partial ADA deficiency. Most of these children carry two different mutant alleles. We were able to correlate genotype and phenotype and to dissect the activity of individual mutant alleles.
我们之前已对7名腺苷脱氨酶（ADA；腺苷氨基水解酶，EC 3.5.4.4）部分缺乏的儿童中的突变酶进行了特征描述。6名儿童有共同的起源，提示有一个共同的祖先。然而，我们发现了多种表型不同的突变酶的证据。我们推测许多突变会发生在CpG二核苷酸处，这是5-甲基胞嘧啶自发脱氨导致C到T或G到A转换的热点。用识别CpG二核苷酸的Msp I和Taq I酶消化这些儿童的DNA，鉴定出三种不同的突变，每种突变都与一种不同突变酶的表达相关。对通过聚合酶链反应扩增的cDNA克隆和基因组DNA进行测序，证实了在CpG二核苷酸处存在C到T或G到A的转换（C226到T、G446到A以及C821到T，分别导致Arg76变为Trp、Arg149变为Gln以及Pro274变为Leu）。在两名ADA缺乏的重症联合免疫缺陷儿童中还偶然发现了一个“无效”突变，表现为获得了一个Msp I位点。同时失去一个Bal I位点确定了精确的碱基替换（T320到C，Leu107变为Pro），序列分析证实了这一点。为了确定通过新生儿筛查项目连续确定的这些儿童中热点突变的真实频率,我们对其余等位基因的cDNA进行了测序。另外两个是热点突变（C631到T和G643到A，分别导致Arg211变为Cys以及Ala215变为Thr），每种突变再次导致一种表型不同的突变酶的表达。只有另外一个突变（之前我们已鉴定出）不在热点区域。这7个突变占了这些儿童所有的14条染色体。因此，在ADA部分缺乏中热点突变的频率非常高。这些儿童中的大多数携带两个不同的突变等位基因。我们能够将基因型与表型相关联，并剖析各个突变等位基因的活性。
