Molecular description of a hypoxanthine phosphoribosyltransferase gene deletion in Lesch-Nyhan syndrome.
Molecular description of a hypoxanthine phosphoribosyltransferase gene deletion in Lesch-Nyhan syndrome.
复制标题
Lesch-Nyhan 综合征中次黄嘌呤磷酸核糖转移酶基因缺失的分子描述。
DOI：
10.1093/hmg/3.1.199
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发表时间：
1994
影响因子：
3.5
通讯作者：
Nelsen,AJ
中科院分区：
文献类型：
--
作者：
Fuscoe,JC;Nelsen,AJ
酶次黄嘌呤磷酸核糖基转移酶（HPRT：EC 2.4. 2.8)催化5 '磷酸核糖基-1-焦磷酸与嘌呤碱基次黄嘌呤或鸟嘌呤缩合形成核苷单磷酸（1）。这种酶的遗传缺陷导致X染色体连锁的Lesch-Nyhan（LN）综合征（2），其特征是高尿酸血症、发育迟缓和舞蹈手足徐动症和自残的神经异常（3）。hprt基因已被克隆（4，S），该基因座的57 kb DNA序列，包括整个hprt基因，已被确定（6）。导致LN综合征的突变已经从聚合酶链反应（PCR）扩增的cDNA（7，8）和个体外显子（9）的DNA序列分析中确定。然而，大约15%的LN突变是由于大的基因组改变，如Southern印迹（10）或PCR（9）分析所揭示的，并且这些突变更难以在分子水平上表征。我们在这里报告的第一个大LN缺失突变的分子分析，包括DNA序列的缺失断点交界处。来自男性Lesch-Nyhan患者的类淋巴母细胞系RJK 984由CT Caskey博士（Baylor College of Medicine，Houston，TX）提供，并具有包括hprt基因外显子6-9的缺失（9）。通过多重hprt PCR确认缺失（9），这将一个缺失断裂点定位到包括内含子S的3100 bp区域。另一个断裂点位于hprt下游一个以前未确定的位置，为了更准确地鉴定断裂点，使用了Southern印迹杂交和PCR。使用含有外显子5的PCR片段作为探针的Southern分析（引物和条件描述于（9））表明断裂接近外显子6。通过PCR分析进一步细化基因内的断裂位点位置。设计引物对（见表1）并用于检测RJK 984 DNA中序列的存在或不存在。用引物A22 S和A226进行的PCR得到预期大小的产物，用引物A22 S和A227进行的PCR也是如此。使用引物A230和A228或A229的PCR不能从RJK 984 DNA产生产物，但确实从野生型DNA扩增出预期大小的片段。PCR反应物由10 mM Tris HCl pH 8.3、1.5 mM MgCl 2、50 mM KCl、0.01%明胶、0.2 mM dNTP和0.3-0.6 μ M的每种引物组成。在94 ℃初始变性4分钟后，反应在94 ℃，1分钟; 59 ℃，1分钟; 72 ℃，0.5-1.5分钟循环32次。这些分析表明断裂位点在紧接外显子6上游的核苷酸34590和34873之间的小于300 bp的区域中（根据Edwards等编号）。（六））。
The enzyme hypoxanthine phosphoribosyltransferase (HPRT: EC 2.4. 2.8) catalyzes the condensation of 5'phosphoribosyl-1-pyrophosphate and the purine bases hypoxanthine or guanine to form nucleoside monophosphates (1). Inherited deficiency of this enzyme results in the X-chromosome linked Lesch-Nyhan (LN) syndrome (2) characterized by hyperuricemia, developmental delay, and the neurological abnormalities of choreoathetosis and self-mutilation (3). The hprt gene has been cloned (4, S) and 57 kb of DNA sequence at the locus, including the entire hprt gene, has been determined (6). Mutations leading to the LN syndrome have been defined from the DNA sequence analysis of polymerase chain reaction (PCR)-amplified cDNA (7, 8) and individuals exons (9). Approximately 15% of LN mutations, however, are due to large genomic alterations as revealed by Southern blot (10) or PCR (9) analysis and these mutations have been more difficult to characterize at the molecular level. We report here the first molecular analysis of a large LN deletion mutation that includes the DNA sequence across the deletion breakpoint junction. Lymphoblastoid line RJK 984, derived from a male Lesch—Nyhan patient, was provided by Dr CT Caskey (Baylor College of Medicine, Houston, TX) and has a deletion which includes exons 6-9 of the hprt gene (9). The deletion was confirmed by multiplex hprt PCR (9) and this located one deletion breakpoint to a 3100 bp region including intron S. The other breakpoint is at a previously undetermined location downstream of hprt.In order to more accurately identify the breaksite, both Southern blot hybridization and PCR were used. Southern analysis using an exon 5-containing PCR fragment as probe (primers and conditions described in (9)) suggested the break was close to exon 6. The breaksite location within the gene was further refined by PCR analysis. Primer-pairs (see Table 1) were designed and used to test for the presence or absence or the sequence in RJK 984 DNA. PCR with primers A22S and A226 gave a product of expected size as did PCR with primers A22S and A227. PCR with primers A230 and A228 or A229 failed to produce a product from RJK 984 DNA but did amplify a fragment of expected size from wild-type DNA. PCR reactions were composed of 10 mM Tris HC1 pH 8.3, 1.5 mM MgCl2, 50 mM KC1, 0.01% gelatin, 0.2 mM dNTPs, and 0.3-0.6/iM of each primer. The reactions were cycled 32 times through 94 C, 1 min; 59 C, 1 min; 72 C, 0.5-1.5 min after an initial denaturation of 94 C for 4 min. These analyses indicated that the breaksite was in a region of less than 300 bp immediately upstream of exon 6 between nucleotides 34590 and 34873 (numbering according to Edwards et al.(6)).