Identification and functional analysis of mutations in the Hypocretin (Orexin) genes of narcoleptic canines
Identification and functional analysis of mutations in the Hypocretin (Orexin) genes of narcoleptic canines
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DOI：
10.1101/gr.gr-1610r
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发表时间：
2001-04-01
期刊：
影响因子：
7
通讯作者：
Mignot, E
中科院分区：
文献类型：
--
作者：
Hungs, M;Fan, J;Mignot, E
嗜睡症是一种影响动物和人类的睡眠障碍。下丘脑分泌素/食欲素受体-2（Hcrtr 2）基因的外显子跳跃突变被确定为杜宾犬和拉布拉多犬发作性睡病的原因。前下丘脑泌素原（Hcrt）基因敲除小鼠具有与人类和犬嗜睡症相似的症状。在这项研究中，11个散发病例的犬发作性睡病和两个额外的多重家庭进行了调查可能的Hcrt和Hcrtr 2突变。散发病例已被证明具有更多变的疾病发作、增加的疾病严重程度和脑脊液中检测不到的下丘脑泌素-1水平。犬Hcrt基因座被分离并表征用于该项目。在这两个位点中仅发现一个新的突变。这种改变导致了一个单一的氨基酸取代（E54 K）在N-末端区域的Hcrtr 2受体和常染色体隐性遗传的腊肠犬家族。还使用用野生型和突变构建体转染的HEK-293细胞系进行先前描述的外显子跳跃突变和E54 K取代的功能分析。结果表明截短的Hcrtr 2蛋白，缺乏适当的膜定位，和不可检测的结合和信号转导外显子跳跃突变的构建体。相反，E54 K异常与适当的膜定位，配体结合的损失，并显着减少钙动员受体的激活。这些结果与所有三种突变的功能丧失一致。在散发病例中没有突变也表明了犬嗜睡症的遗传异质性，正如以前在人类中报道的那样。
Narcolepsy is a sleep disorder affecting animals and humans. Exon skipping mutations of the Hypocretin/Orexin-receptor-2 (Hcrtr2) gene were identified as the cause of narcolepsy in Dobermans and Labradors. Preprohypocretin (Hcrt) knockout mice have symptoms similar to human and canine narcolepsy. In this study, 11 sporadic cases of canine narcolepsy and two additional multiplex families were investigated for possible Hcrt and Hcrtr2 mutations. Sporadic cases have been shown to have more variable disease onset, increased disease severity, and undetectable Hypocretin-1 levels in cerebrospinal fluid. The canine Hcrt locus was isolated and characterized for this project. Only one novel mutation was identified in these two loci. This alteration results in a single amino acid substitution (E54K) in the N-terminal region of the Hcrtr2 receptor and autosomal recessive transmission in a Dachshund family. Functional analysis of previously-described exon-skipping mutations and of the E54K substitution were also performed using HEK-293 cell lines transfected with wild-type and mutated constructs. Results indicate a truncated Hcrtr2 protein, an absence of proper membrane localization, and undetectable binding and signal transduction for exon-skipping mutated constructs. In contrast, the E54K abnormality was associated with proper membrane localization, loss of ligand binding, and dramatically diminished calcium mobilization on activation of the receptor. These results are consistent with a loss of function for all three mutations. The absence of mutation in sporadic cases also indicates genetic heterogeneity in canine narcolepsy, as reported previously in humans.