Identification of a common nonsense mutation in Japanese patients with type I adenine phosphoribosyltransferase deficiency.
Identification of a common nonsense mutation in Japanese patients with type I adenine phosphoribosyltransferase deficiency.
复制标题
鉴定日本 I 型腺嘌呤磷酸核糖转移酶缺乏症患者中常见的无义突变。
DOI：
10.1093/nar/18.19.5915
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发表时间：
1990
影响因子：
14.9
通讯作者：
Tischfield,JA
中科院分区：
文献类型：
--
作者：
Sahota,A;Chen,J;Asaki,K;Takeuchi,H;Stambrook,PJ;Tischfield,JA
在人体缺乏腺嘌呤磷酸核糖基转移酶（APRT）活性的情况下，腺嘌呤被黄嘌呤氧化酶氧化为高度不溶性和肾毒性的衍生物2,8 -二羟基腺嘌呤（DHA）。APRT缺乏的两种表型变异已被确认(1)。I型缺乏症（完全酶缺乏症）已在许多不同的国家发现，但H型缺乏症（体内完全缺乏症，但细胞提取物部分缺乏症）仅在日本发现。II型缺乏具有共同的祖先起源，是日本人形成DHA结石的主要原因（约75%）(2)。大多数H型患者外显子5的错义突变和内含子2/2 TaqI RFLP是纯合的(3)，但他们在远端SphI RFLP位点上表现出两种不同的模式(4)。H型突变等位基因命名为APRT* J(2)。两个患有I型缺陷的非日本兄弟是APRT* QO零等位基因的复合杂合子(5)，但他们是1/1 TaqI模式的同质(6)。我们分析了两名日本患者的突变分子性质，一名是来自近亲婚姻的13岁I型男性（ASA1），另一名是表现为H型表型但有一个APRT* j型和一个APRT* qo型等位基因的16岁男性（TAK2） （Sahota et al.，提交）。ASAl显示1/1 TaqI RFLP，而TAK2显示1/2模式。ASAI在淋巴母细胞提取物中没有检测到APRT活性，但TAK2在我们的对照范围内（584-633 nmol/h/mg）具有25%的活性。两名患者的培养淋巴细胞均未显示功能性APRT活性，这表明：(i)完全抵抗2,6 -二氨基嘌呤的生长抑制；（ii）不能吸收外源性腺嘌呤；（iii）在腺嘌呤-氮杂塞嘧啶-丙氨酸培养基中缺乏生长。从培养的淋巴细胞中分离到基因组dna，通过聚合酶链反应（PCR）扩增出包含APRT基因编码区和侧翼序列的2.4 kb片段。将PCR产物亚克隆到M13中，对每个患者的3个阳性克隆进行完全测序，以确定一致的基因序列并纠正PCR错误。在ASAl中发现了碱基1449的A-to-T翻转。这种突变导致trp98 （TGG）被3号外显子的无义密码子（TGA）取代。在1453位点（GCC到GCT， alagg）也发现了一个沉默的碱基取代。此外，在1184位（内含子2）和1651位（内含子3）分别观察到C-to-T和G-to-A的转变。对于我们的野生型序列，ATG起始密码子的A被指定为1 （Chen et al.，提交）。通过对PCR DNA的直接测序和TaqI酶切，证实了ASAl中98和99个氨基酸位置的碱基替换、内含子多态性和TaqI多态性位点的纯合性。在TAK2患者的APRT* QO等位基因中发现了相同的突变和多态性。该患者的另一个等位基因为APRT* J型，包括2/2 TaqI RFLP模式。该患者的APRT* J等位基因在1651位也出现G-to-A替换。我们已经在所有日本和非日本aprt缺陷患者的DNA中观察到这种碱基变化，这表明我们的野生型序列在这个位置上是罕见的变异。在ASAI和TAK2的APRT* QO等位基因中发现的外显子3突变和沉默替代也在其他三名日本I型患者中发现，并且在其中至少一名患者中发现了内含子多态性(7)。这种无意义的突变在10个非…
In the absence of adenine phosphoribosyltransferase (APRT) activity in man, adenine is oxidized by xanthine oxidase to the highly insoluble and nephrotoxic derivative, 2, 8-dihydroxyadenine (DHA). Two phenotypic variants of APRT deficiency have been recognized (1). Type I deficiency (complete enzyme deficiency) has been observed in many different countries, but type H deficiency (complete deficiency invivo but partial deficiency in cell extracts) is found only in Japan. Type II deficiency has a common ancestral origin and is the main cause (about 75%) of DHA stone formation in the Japanese (2). The majority of Type H patients is homozygous for a missense mutation in exon five and for the 2/2 TaqI RFLP in intron two (3), but they show two different patterns with respect to a remote SphI RFLP site (4). The Type H mutant alleles are designated APRT* J (2). Two non-Japanese brothers with Type I deficiency are compound heterozygotes for APRT* QO null alleles (5), but they are homogygous for the 1/1 TaqI pattern (6). We have analyzed the molecular nature of the mutation in two Japanese patients, a thirteen-year-old Type I male (ASA1) from a consanguineous marriage, and a sixteen-year-old male (TAK2) who exhibits the Type H phenotype but has one APRT* J-type and one APRT* QO-type allele (Sahota et al., submitted). ASAl displays the 1/1 TaqI RFLP whereas TAK2 displays the 1/2 pattern. ASAI has no detectable APRT activity in lymphoblast extracts, but TAK2 has 25% of the activity ofour control range (584-633 nmol/h/mg). Cultured lymphoblasts from neither patient showed functional APRT activity as indicated by:(i) complete resistance to growth inhibition by 2, 6-diaminopurine;(ii) inability to take up exogenous adenine; and (iii) lack ofgrowth in adenine-azaserine-alanosine medium. Genomic DNAwas isolated from cultured lymphoblasts and a 2.4 kb fragment containing the coding region and the flanking sequences of the APRT gene amplified by the polymerase chain reaction (PCR). The PCR product was subcloned into M13 and three positive clones from each patient were completely sequenced to determine the consensus gene sequence and to correct for PCR errors. An A-to-T transversion at base 1449 was identified in ASAl. This mutation leads to the replacement of trp98 (TGG) by a nonsense codon (TGA) in exon three. A silent base substitution at position 1453 (GCC to GCT, alagg) was also found. In addition, C-to-T and G-to-A transitions were observed at positions 1184 (intron two) and 1651 (intron three), respectively. The A of the ATG start codon is designated 1 with respect to our wild-type sequence (Chen et al., submitted). The homozygous nature of the base substitutions at amino acid positions 98 and 99, intron polymorphisms, and the TaqI polymorphic site in ASAl were confirmed by direct sequencing and TaqI digestion of PCR DNA. The same mutation and polymorphisms were found in the APRT* QO allele from patient TAK2. The other allele in this patient was of the APRT* J type, including the 2/2 TaqI RFLP pattern. The APRT* J allele from this patient also showed the G-to-A substitution at position 1651. We have observed this base change in the DNA from all the Japanese and non-Japanese APRT-deficient patients we have sequenced, suggesting that our wild-type sequence is a rare variant at this position.The exon three mutation and the silent substitution identified in the APRT* QO allele from ASAI and TAK2 have also been found in three other Type I Japanese patients, and the intron polymorphisms have been identified in at least one of these patients (7). This nonsense mutation has not been observed in ten non …