一个睑裂狭小-倒转型内眦赘皮-上睑下垂综合征家系FOXL2基因突变筛查及蛋白质构象分析

FOXL2 gene mutation screening and protein conformation analysis in a family with blepharophimosis-ptosis-epicanthus inversus syndrome

目的研究一个BPES家系进行FOXL2基因的突变分析,探讨BPES发生的分子机制。方法纳入一个先天性BPES大家系和10例正常对照者。首先抽取研究对象外周静脉血2 ml并抽提基因组DNA,然后用四组相互重叠的引物通过聚合酶链反应扩增FOXL2基因的整个编码区和附近的内含子序列并测序。最后用DNAstar软件分析测序结果,ExPASy和Predictprotein软件对比分析突变前后的蛋白质构象。结果这个家族的所有患者都表现出典型的BPES II型特征,包括睑裂狭小、上睑下垂、倒转型内眦赘皮,没有女性卵巢早衰(POF)。在家系所有BPES患者中均发现C.749~778 dup 30的重复突变,而家系中的正常人及10例正常对照者均未发现FOXL2基因突变。蛋白质构象分析示C.749~778 dup 30突变引起翻译后蛋白质增加了10个氨基酸,蛋白质分子量和等电点均升高,α-螺旋所占的比例增加了0.6%,β折叠和无规卷曲所占比例相应下降。结论在BPES家系中发现了一个新的FOXL2基因突变位点,扩大了FOXL2基因突变谱的范围,并为FOXL2蛋白提供了新的的结构功能见解。

Objective The purpose of this study was to explore the molecular mechanism of BPES by FOXL2 gene mutation analysis in a Chinese family with BPES.Methods A large congenital autosomal dominant Chinese BPES family and 10 normal controls were enrolled in this study.DNA was obtained from the leukocytes in peripheral venous blood.And then the entire coding region and nearby intron sequences of the FOXL2 gene were amplified by polymerase chain reaction(PCR)with four overlapping sets of primers and sequencing analyses.The sequencing results were analyzed using the DNAstar software,and the protein conformation before and after mutation was compared using the ExPASy and Predictprotein software.Results All patients of the family demonstrated typical features of type II BPES,including narrow palpebral fissures,ptosis,telecanthus,and epicanthus inversus without premature ovarian failure(POF).A novel FOXL2 duplicate mutation C.749~778 dup 30,including a 30-bp duplication,which would result in an insertion of 10 additional amino acids,was identified in all affected family members,while the FOXL2 gene mutation was not found in any normal controls.The protein conformation analysis showed that the C.749~778 dup 30 increased the protein molecular weight and isoelectric point by 10 amino acids,the proportion ofα-helix increased by 0.6%,and the proportion ofβfolding and aperiodical coil decreased accordingly.Conclusions A novel mutation site of FOXL2 gene was identified in a Chinese family with BPES,which expanded the mutation spectrum of FOXL2 gene and provided additional structural and functional insights for FOXL2 proteins.