Abstract：
OBJECTIVE: This study investigated the new splice site mutations of Myosin VIIA (MYO7A) in patients with Usher syndrome type 1 (USH1) from a three-generation Chinese consanguineous family.
METHODS: All subjects underwent comprehensive ophthalmic examinations and an audiometric test. Demographic data, family history, and peripheral blood leukocytes were collected. We performed whole exome sequencing (WES) to analyze the genomic DNA of the family. DNA sequence and restriction fragment length polymorphism (RFLP) analyses were also done. The identified genetic variants were validated by conducting polymerase chain reaction (PCR) in 100 healthy control subjects and comparing with the NCBI VARIANT database and the 1000 Genomes Project. The functional consequences were further analyzed.
RESULTS: WES identified two new splice site mutations (c.5648G > A(rs111033215) and c.6238-1G > C) in MYO7A in two patients with USH1, i.e., the proband and her elder brother. DNA sequence and RFLP analyses showed that other members without USH1 carried only one of the two mutations. In the analysis of healthy controls, neither mutation existed. Both mutations were predicted to be damaging and were most likely associated with USH1.
CONCLUSIONS: In the three-generation Chinese consanguineous family with USH1, c.5648G > A(rs111033215) and c.6238-1G > C mutations in MYO7A are most likely associated with the disease. Our findings expand the mutational spectrum of MYO7A, which will enhance the understanding of the genetic abnormalities in USH1 and provide more evidence for future investigations on therapeutic strategies such as precise gene replacement or gene editing.
METHODS: All subjects underwent comprehensive ophthalmic examinations and an audiometric test. Demographic data, family history, and peripheral blood leukocytes were collected. We performed whole exome sequencing (WES) to analyze the genomic DNA of the family. DNA sequence and restriction fragment length polymorphism (RFLP) analyses were also done. The identified genetic variants were validated by conducting polymerase chain reaction (PCR) in 100 healthy control subjects and comparing with the NCBI VARIANT database and the 1000 Genomes Project. The functional consequences were further analyzed.
RESULTS: WES identified two new splice site mutations (c.5648G > A(rs111033215) and c.6238-1G > C) in MYO7A in two patients with USH1, i.e., the proband and her elder brother. DNA sequence and RFLP analyses showed that other members without USH1 carried only one of the two mutations. In the analysis of healthy controls, neither mutation existed. Both mutations were predicted to be damaging and were most likely associated with USH1.
CONCLUSIONS: In the three-generation Chinese consanguineous family with USH1, c.5648G > A(rs111033215) and c.6238-1G > C mutations in MYO7A are most likely associated with the disease. Our findings expand the mutational spectrum of MYO7A, which will enhance the understanding of the genetic abnormalities in USH1 and provide more evidence for future investigations on therapeutic strategies such as precise gene replacement or gene editing.
摘要：
目的：这项研究调查了来自中国三代近亲家族的Usher综合征1型（USH1）患者肌球蛋白VIIA（MYO7A）的新剪接位点突变。
方法：所有受试者都接受了全面的眼科检查和听力测试。人口统计数据,家族史,收集外周血白细胞。我们进行了全外显子组测序（WES）以分析该家族的基因组DNA。还进行了DNA序列和限制性片段长度多态性（RFLP）分析。通过在100名健康对照受试者中进行聚合酶链反应（PCR）并与NCBIVARIANT数据库和1000基因组项目进行比较来验证所鉴定的遗传变异。进一步剖析了机能后果。
结果：WES在两名USH1患者的MYO7A中发现了两个新的剪接位点突变（c.5648G>A（rs111033215）和c.6238-1G>C），即先证者和她哥哥.DNA序列和RFLP分析表明，没有USH1的其他成员仅携带两个突变之一。在健康对照的分析中，两种突变都不存在。两种突变都被预测为破坏性的，并且很可能与USH1相关。
结论：在具有USH1的三代中国近亲家族中，MYO7A中c.5648G>A（rs111033215）和c.6238-1G>C突变最可能与该疾病相关。我们的发现扩展了MYO7A的突变谱，这将增强对USH1中遗传异常的理解，并为未来对治疗策略的研究提供更多证据，例如精确的基因替换或基因编辑。
方法：所有受试者都接受了全面的眼科检查和听力测试。人口统计数据,家族史,收集外周血白细胞。我们进行了全外显子组测序（WES）以分析该家族的基因组DNA。还进行了DNA序列和限制性片段长度多态性（RFLP）分析。通过在100名健康对照受试者中进行聚合酶链反应（PCR）并与NCBIVARIANT数据库和1000基因组项目进行比较来验证所鉴定的遗传变异。进一步剖析了机能后果。
结果：WES在两名USH1患者的MYO7A中发现了两个新的剪接位点突变（c.5648G>A（rs111033215）和c.6238-1G>C），即先证者和她哥哥.DNA序列和RFLP分析表明，没有USH1的其他成员仅携带两个突变之一。在健康对照的分析中，两种突变都不存在。两种突变都被预测为破坏性的，并且很可能与USH1相关。
结论：在具有USH1的三代中国近亲家族中，MYO7A中c.5648G>A（rs111033215）和c.6238-1G>C突变最可能与该疾病相关。我们的发现扩展了MYO7A的突变谱，这将增强对USH1中遗传异常的理解，并为未来对治疗策略的研究提供更多证据，例如精确的基因替换或基因编辑。
