Congenital Hypothyroidism Due to Truncating PAX8 Mutations: A Case Series and Molecular Function Studies
Congenital Hypothyroidism Due to Truncating PAX8 Mutations: A Case Series and Molecular Function Studies
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DOI：
10.1210/clinem/dgaa584
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发表时间：
2020-11-01
影响因子：
5.8
通讯作者：
Narumi, Satoshi
中科院分区：
文献类型：
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作者：
Iwahashi-Odano, Megumi;Nagasaki, Keisuke;Narumi, Satoshi
背景：PAX8是甲状腺发育所需的转录因子，其突变可导致先天性甲状腺功能减退症（CH）。已有超过20个实验验证的PAX8功能丧失突变被描述，除了一个之外，所有突变都位于dna结合配对结构域。目的：我们报道了位于配对结构域外的3个新的截断PAX8突变的鉴定和功能表征。方法：在新生儿筛查框架中诊断为甲状腺发育不全的3例CH先证患者给予左旋甲状腺素治疗。进行基于下一代测序的突变筛选。鉴定的突变的功能通过Western blotting、细胞内定位测定和HeLa细胞的反激活测定来验证。荧光素酶互补法用于评估突变对PAX8与其伴侣蛋白NKX2-1相互作用的影响。结果：每个先证都有新的截断PAX8突变，分别为I160Sfs*52、Q213Efs*27和F342Rfs*85。Western blotting显示I160fs-PAX8蛋白不稳定。Q213fs-PAX8和F342fs-PAX8蛋白表达水平正常，核定位正常，但荧光素酶报告基因失活。通过荧光素酶互补实验，我们发现PAX8-NKX2-1相互作用在Q213fs-PAX8中存在缺陷。我们还对重组PAX8蛋白进行了表征，发现外显子10对应的蛋白序列（363-400个残基）是PAX8- nkx2 -1相互作用所必需的。结论：报告了3个位于配对结构域外的新型截断PAX8突变的临床和分子发现。利用培养细胞和重组蛋白进行的实验表明，PAX8的c端部分（即363- 400aa）是PAX8- nkx2 -1相互作用所必需的。
Context: PAX8 is a transcription factor required for thyroid development, and its mutation causes congenital hypothyroidism (CH). More than 20 experimentally verified loss-of-function PAX8 mutations have been described, and all but one were located in the DNA-binding paired domain.Objective: We report the identification and functional characterization of 3 novel truncating PAX8 mutations located outside the paired domain.Methods: Three CH probands, diagnosed in the frame of newborn screening, had thyroid hypoplasia and were treated with levothyroxine. Next-generation sequencing-based mutation screening was performed. Functionality of the identified mutations were verified with Western blotting, intracellular localization assays, and transactivation assays with use of HeLa cells. Luciferase complementation assays were used to evaluate the effect of mutations on the interaction between PAX8 and its partner, NKX2-1.Results: Each proband had novel truncating PAX8 mutations that were I160Sfs*52, Q213Efs*27, and F342Rfs*85. Western blotting showed destabilization of the I160fs-PAX8 protein. Q213fs-PAX8 and F342fs-PAX8 showed normal protein expression levels and normal nuclear localization, but showed loss of transactivation of the luciferase reporter. By luciferase complementation assays, we showed that PAX8-NKX2-1 interaction was defective in Q213fs-PAX8. We also characterized the recombinant PAX8 proteins, and found that the protein sequence corresponding to exon 10 (363-400 as residues) was essential for the PAX8-NKX2-1 interaction.Conclusions: Clinical and molecular findings of 3 novel truncating PAX8 mutations located outside the paired domain were reported. Experiments using cultured cells and recombinant proteins showed that the C-terminal portion (ie, 363-400 aa) of PAX8 is required for the PAX8-NKX2-1 interaction.