Kishnani P S, Bao Y, Wu J Y, Brix A E, Lin J L, Chen Y T
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochem Mol Med. 1997 Aug;61(2):168-77. doi: 10.1006/bmme.1997.2600.
Two Maltese puppies with massive hepatomegaly and failure to thrive had isolated deficient glucose-6-phosphatase (G-6-Pase) activity in liver and kidney and pathological findings compatible with GSD-Ia. To identify the mutation, we cloned G-6-Pase canine cDNA by RT-PCR with primers from the murine G-6-Pase gene sequence. The canine G-6-Pase cDNA is 2346 bp, with a 5' untranslated region of 87 bp, a coding region of 1071 bp, and a 3' untranslated region of 1185 bp. The difference between the canine and human sequences is in the 3' untranslated region. A greater than 90% amino acid sequence homology was seen with canine, human, murine, and rat G-6-Pase. G-6-Pase cDNA from affected and control puppies revealed complete homology except at nt position 450, which showed a guanine to cytosine (G to C) transversion resulting in substitution of a methionine by isoleucine at codon 121 (M121I) in all five clones studied. The loss of an NcoI restriction site on genomic DNA amplified with primers flanking the mutation allowed us to prove that affected puppies were homozygous for the mutation and parents were heterozygous carriers. The mutant G-6-Pase cDNA had 15 times less enzyme activity than wild-type cDNA following transient transfection. Northern blot analysis of puppies with GSD-Ia revealed increased G-6-Pase mRNA, compared to normal controls. Increased G-6-Pase mRNA was also seen in normal fasted puppies compared to littermates in the fed state, suggesting that the increased G-6-Pase mRNA is a physiologic response to fasting. This is the first report of a molecularly confirmed naturally occurring animal model of GSD-Ia. The establishment of a breeding colony of this dog strain will facilitate studies on the role of G-6-Pase gene in glucose homeostasis, in pathophysiology of disease, and development of novel therapeutic approaches such as gene therapy.
两只患有严重肝肿大且发育不良的马耳他幼犬，其肝脏和肾脏中葡萄糖-6-磷酸酶（G-6-Pase）活性单独缺乏，病理检查结果与I型糖原贮积病（GSD-Ia）相符。为了鉴定该突变，我们使用来自小鼠G-6-Pase基因序列的引物通过逆转录聚合酶链反应（RT-PCR）克隆了犬G-6-Pase cDNA。犬G-6-Pase cDNA为2346 bp，5'非翻译区为87 bp，编码区为1071 bp，3'非翻译区为1185 bp。犬和人序列之间的差异在于3'非翻译区。犬、人、小鼠和大鼠的G-6-Pase氨基酸序列同源性超过90%。来自患病和对照幼犬的G-6-Pase cDNA除了在核苷酸位置450处显示出鸟嘌呤到胞嘧啶（G到C）的颠换外，其余完全同源，这导致在所有五个研究克隆的第121密码子（M121I）处甲硫氨酸被异亮氨酸取代。用突变侧翼引物扩增的基因组DNA上NcoI限制性酶切位点的缺失使我们能够证明患病幼犬为该突变的纯合子，而父母为杂合子携带者。瞬时转染后，突变型G-6-Pase cDNA的酶活性比野生型cDNA低15倍。对患有GSD-Ia的幼犬进行Northern印迹分析发现，与正常对照相比，G-6-Pase mRNA增加。与处于进食状态的同窝幼犬相比，正常禁食幼犬中也观察到G-6-Pase mRNA增加，这表明G-6-Pase mRNA增加是对禁食的生理反应。这是第一份分子证实的GSD-Ia自然发生动物模型的报告。建立这种犬种的繁殖群体将有助于研究G-6-Pase基因在葡萄糖稳态、疾病病理生理学中的作用以及开发诸如基因治疗等新的治疗方法。
