Shi Xi, Zhang Yan, Qiu Shiwei, Zhuang Wei, Yuan Na, Sun Tiantian, Gao Jian, Qiao Yuehua, Liu Ke
The Institute of Audiology and Balance science of Xuzhou Medical University, Xuzhou, China.
Department of Otolaryngology-Head and Neck Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
J Clin Lab Anal. 2018 Sep;32(7):e22444. doi: 10.1002/jcla.22444. Epub 2018 Apr 17.
To investigate whether a novel compound heterozygous mutations c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) in GJB2 result in hearing loss.
Allele-specific PCR-based universal array (ASPUA) screening and sequence analysis were applied to identify these mutations. 3D model was built to perform molecular dynamics (MD) simulation to verify the susceptibility of the mutations. Furthermore, WT- and Mut-GJB2 DNA fragments, containing the mutation of c.257C>G and c.176del16 were respectively cloned and transfected into HEK293 and spiral ganglion neuron cell (SGNs) by lenti-virus delivery system to indicate the subcellular localization of the WT- and Mut-CX26 protein.
A novel compound heterozygous mutation c.257C>G (p.T86R)/c.176del16 (p.G59A fs18) in GJB2 was identified in a Chinese family, in which 4 siblings with profound hearing loss, but the fifth child is normal. By ASPUA screening and sequencing, a compound heterozygote mutations in GJB2 c.257C>G (p.T86R)/c.176del16 (p.G59A fs18) were identified in these four deaf children, each of the mutated GJB2 gene were inherited from their parents. There is no mutation of GJB2 gene identified in the normal child. Besides, the compound heterozygous mutation GJB2 c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) could lead to the alterations of the subcellular localization of each corresponding mutated CX26 protein and could cause the hearing loss, which has been predicted by MD simulation and verified in both 293T and SGNs cell line.
The c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) compound mutations in GJB2 detected in this study are novel, and which may be associated with hearing loss in this Chinese family.
研究GJB2基因中一种新的复合杂合突变c.257C>G（p.T86R）/c.176del16（p.G59A fs*18）是否导致听力损失。
应用基于等位基因特异性PCR的通用阵列（ASPUA）筛选和序列分析来鉴定这些突变。构建三维模型进行分子动力学（MD）模拟以验证突变的易感性。此外，分别克隆含有c.257C>G和c.176del16突变的野生型和突变型GJB2 DNA片段，并通过慢病毒递送系统转染到HEK293和螺旋神经节神经元细胞（SGNs）中，以显示野生型和突变型CX26蛋白的亚细胞定位。
在中国一个家庭中鉴定出GJB2基因中的一种新的复合杂合突变c.257C>G（p.T86R）/c.176del16（p.G59A fs18），其中4个兄弟姐妹患有重度听力损失，但第五个孩子正常。通过ASPUA筛选和测序，在这4名聋儿中鉴定出GJB2基因的复合杂合突变c.257C>G（p.T86R）/c.176del16（p.G59A fs18），每个突变的GJB2基因均遗传自其父母。在正常孩子中未鉴定出GJB2基因突变。此外，复合杂合突变GJB2 c.257C>G（p.T86R）/c.176del16（p.G59A fs*18）可导致每个相应突变的CX26蛋白的亚细胞定位改变，并可导致听力损失，这已通过MD模拟预测并在293T和SGNs细胞系中得到验证。
本研究中检测到的GJB2基因中的c.257C>G（p.T86R）/c.176del16（p.G59A fs*18）复合突变是新的，并且可能与这个中国家庭中的听力损失有关。
