视锥细胞功能异常眼病的致病基因研究和全色盲小鼠模型的基因治疗

发布时间：2018-09-09 19:04

【摘要】：目的：Alstrom综合征(Alstrom syndrome, AS)是一种少见的常染色体隐性遗传病,临床表现时视锥视杆细胞营养不良和多器官功能障碍。目前唯一已知的致病基因ALMS1基因突变可引起Alstrom综合征。本研究的目的是鉴定Alstrom综合征患者的致病突变并评价该综合征的临床特点。方法：收集患者病史,进行眼部和与该疾病相关的全身检查,并采集患者及其父母静脉血,提取基因组DNA。对致病基因ALMS1的外显子、外显子和内含子交界区序列进行PCR (polymerase chain reaction, PCR1扩增,目的产物切胶纯化后直接测序。与基因库中ALMS1的转录本(NM015120.4)进行比对,排除单核苷酸多态(single nucleotide polymorphism, SNP)并确定致病突变。结果：共有来自5个家系的7名患者诊断为Alstrom综合征,并且这些家系都是非近亲家系。这些患者均有视锥视杆细胞营养不良的临床表现,视力差,畏光和眼球震颤。另外患者还伴有感音性耳聋、肥胖、胰岛素抵抗、2型糖尿病、肝肾功能障碍、高胰岛素血症、甲状腺功能减退、智力障碍、黑棘皮症和脊柱侧凸等其他临床症状。基因检测结果发现：患者1的两个新移码突变p.N3150Kfs2X和p.V3154Xfs;患者2的新移码突变p.N3672Ifs11X和曾经报道过的错义突变p.R3703X;患者3的新错义突变和移码突变p.S2479X和p.R3611Efs7X;患者4和5的未曾报道的纯合错义突变p.S695X;患者6和7的新移码突变p.H688HfsX和p.Q3147Qfs2X。在100个无关的健康对照人群中没有发现这些突变。共分离分析显示父母分别是这些突变等位基因的杂合携带者。结论：本研究中7名患者全部都具有Alstrom综合征典型的眼部异常和多系统异常。这些ALMS1的突变导致翻译错误的截短的蛋白不能发挥正常功能,是该综合征发病的遗传基础。目的：研究全色盲患者的临床表现的特征并确定致病基因突变。方法：分别来自于10个家系的15名全色盲患者进行分析。收集患者病史,进行眼部相关检查包括BCVA、色觉、裂隙灯检查、眼底检查、视网膜电流图检查、视野检查和黄斑部的SD-OCT检查。采集患者及其父母静脉血,提取基因组DNA。对致病基因CNGA3、CNGB3、GNAT2、PDE6C和PDE6H的外显子、外显子和内含子交界区进行PCR扩增,目的产物切胶纯化后直接测序。运用Blat tool工具与基因库中标准序列进行比对,排除SNP并确定致病突变,并在家系里进行共分离分析验证突变。突变结果在1000genomes进行比对排除SNP。结果：患者均表现为畏光、眼球震颤和辨色力完全丧失或残存部分辨色能力。患者的最佳矫正视力在0.05～0.2。视锥细胞ERG反应未记录到波形或振幅显著降低,SD-OCT示IS/OS层不同程度消失和黄斑区视网膜厚度变薄。共有8个家系13名患者均发现CNGA3突变。这些突变包括7个新的错义突变和3个新的缺失突变和4个曾经报导过的错义突变。另外2名患者没有检测到5个已知致病基因的任何突变。结论：CNGA3是中国全色盲患者最常见的致病基因。本研究发现10个新的CNGA3突变。发现全色盲患者的遗传学特点对于遗传咨询和将来的基因治疗都很关键。本研究是目前关于中国全色盲患者的首次遗传学报道。目的：对上一部分研究所确定的CNGA3新发纯合错义突变进行初步的体外突变蛋白功能研究。方法：针对CNGA3的纯合错义突变D211E的蛋白功能研究,构建突变型CNGA3基因真核表达载体。在野生型CNGA3基因真核表达载体CNGA3-pCMV6的基础上,利用定点诱变技术,构建D211E突变型CNGA3基因真核表达载体。体外培养HEK293细胞,将野生型以及突变型CNGA3基因真核表达载体CNGA3-pCMV6转染至HEK293细胞,经免疫荧光染色,荧光显微镜观察比较野生型以及突变型CNGA3蛋白在人胚肾细胞(HEK293)中的表达和分布。结果：成功构建突变型CNGA3基因真核表达载体,体外转染至HEK293细胞后,野生型与突变型蛋白分布差异很大。荧光显微镜证实野生型CNGA3蛋白主要分布于细胞膜,在细胞胞浆内仅有少量呈点状、散在分布,而突变型CNGA3蛋白则明显聚集在一起堆积于胞浆中。结论：通过对本研究中所确定的CNGA3基因新发错义突变D211E进行突变蛋白功能研究,提示该突变会明显影响蛋白在细胞中的分布,失去功能的蛋白质是引起发病的分子机制。目的：探讨AAV5载体介导的基因治疗能否恢复全色盲小鼠模型Cnga3Cldfl5的视锥细胞功能。方法：对出生后21天的Cnga3cpfl5进行视网膜下腔注射AAV5-IRBP/GNAT2-hCNGA3载体,注射后视网膜脱离达80%以上并且没有并发症的小鼠用于后续的实验观察。小鼠只接受注射单侧眼,另一眼用作对照观察。注射载体5-6月时记录视网膜电流图。小鼠麻醉颈椎脱臼处死后,将眼球包埋进行冰冻切片。用视蛋白抗体和CNGA3抗体作为一抗,免疫荧光检测视网膜的视蛋白和CNGA3蛋白是否表达。结果：和未注射眼相比较,经过基因治疗视锥细胞的功能得到了明显的恢复。Cnga3Cpfl5小鼠视网膜视锥细胞外节检测到了CNGA3和视蛋白的表达,并且治疗有效的时间至少维持了5-6个月。结论：本研究是在生后3周进行基因治疗的,而且使自然发生的全色盲小鼠Cnga3cpfl5小鼠视锥细胞功能得到了一定程度的恢复。该研究结果对于人类CNGA3突变引起的全色盲的基因治疗有一定的启示作用。
[Abstract]:AIM: Alstrom syndrome (AS) is a rare autosomal recessive inherited disease characterized by cone rod dystrophy and multiple organ dysfunction. The only known mutation in the ALMS1 gene can cause Alstrom syndrome. The clinical characteristics of the syndrome were evaluated.
METHODS: The patient's history was collected, the eye and related systemic examinations were performed, and the venous blood of the patient and his parents was collected to extract genomic DNA. The exon, exon and intron boundary regions of the pathogenic gene ALMS1 were sequenced by polymerase chain reaction (PCR 1). Single nucleotide polymorphism (SNP) was excluded and pathogenic mutations were identified by comparing the transcripts of ALMS1 in the library (NM015120.4).
Results: A total of seven patients from five families were diagnosed with Alstrom syndrome and all of these families were non-inbred. All patients had clinical manifestations of cone rod cell dystrophy, poor vision, photophobia and nystagmus. In addition, the patients were accompanied by sensorineural hearing loss, obesity, insulin resistance, type 2 diabetes mellitus, liver and kidney dysfunction. Other clinical symptoms, such as hyperinsulinemia, hypothyroidism, mental retardation, acanthosis nigricans and scoliosis, were found. Two new frameshift mutations, P. N3150Kfs2X and P. V3154Xfs, P. N3672Ifs11X and the previously reported missense mutation P. R3703X, were detected in patient 1, P. N3150Kfs2X and P. V3154Xfs, respectively. Code mutations p.S2479X and p.R3611Efs7X, homozygous missense mutations p.S695X in patients 4 and 5, and new frameshift mutations p.H688HfsX and p.Q3147Qfs2X in patients 6 and 7 were not found in 100 unrelated healthy controls. Segregation analysis revealed that parents were heterozygous carriers of these mutant alleles, respectively.
CONCLUSION: All 7 patients in this study had typical ocular and multisystem abnormalities in Alstrom syndrome. These mutations in ALMS1 resulted in the inability of truncated proteins with erroneous translations to function properly, which is the genetic basis for the onset of Alstrom syndrome.
Objective: To study the clinical characteristics of patients with achromatic blindness and identify the mutation of the disease causing gene.
Methods: 15 patients with panchromatic blindness from 10 families were analyzed. The patient's history was collected, and the relevant ophthalmic examinations including BCVA, color vision, slit lamp examination, fundus examination, electroretinogram, visual field examination and SD-OCT examination of macula were performed. The blood samples of patients and their parents were collected and genomic DNA was extracted. The exon, exon and intron boundaries of CNGB3, GNAT2, PDE6C and PDE6H were amplified by PCR. The product was purified and sequenced directly. The standard sequence in the gene library was compared with the Blat tool tool to exclude SNP and identify the pathogenic mutation. The mutation was verified by co-isolation and analysis in a family. Comparison and exclusion of SNP.
Results: All patients presented with photophobia, nystagmus and complete loss of color discrimination or residual color discrimination. The best corrected visual acuity of the patients ranged from 0.05 to 0.2. ERG response of cone cells did not show a significant decrease in waveform or amplitude. SD-OCT showed that IS/OS layer disappeared and retinal thickness in macular region became thinner in different degrees. CNGA3 mutations were found. These mutations included seven new missense mutations and three new deletion mutations and four previously reported missense mutations. No mutations in five known pathogenic genes were detected in the other two patients.
CONCLUSION: CNGA3 is the most common pathogenic gene in Chinese patients with panchromatic blindness. Ten new CNGA3 mutations have been found in this study.
AIM: To study the function of a newly purified missense mutation of CNGA3 in vitro.
Methods: The eukaryotic expression vector of mutant CNGA3 gene was constructed based on the homozygous missense mutation D211E of CNGA3. The eukaryotic expression vector of wild-type CNGA3 gene CNGA3-pCMV6 was constructed by site-directed mutagenesis. The wild-type CNGA3 gene and its protrusion were cultured in HEK293 cells in vitro. The expression and distribution of wild-type and mutant CNGA3 proteins in human embryonic kidney cells (HEK293) were observed by immunofluorescence staining and fluorescence microscopy.
Results: The eukaryotic expression vector of mutant CNGA3 gene was successfully constructed and transfected into HEK293 cells in vitro. The distribution of wild-type and mutant CNGA3 proteins was very different. Fluorescence microscopy confirmed that wild-type CNGA3 proteins were mainly distributed in the cell membrane, only a small amount of dot-like and scattered in the cytoplasm, while mutant CNGA3 proteins were obviously concentrated in the cell membrane. It accumulates in cytoplasm.
CONCLUSION: The function of the new missense mutation D211E in CNGA3 gene identified in this study suggests that the mutation can significantly affect the protein distribution in the cells, and the dysfunctional protein is the molecular mechanism of the pathogenesis.
AIM: To investigate whether gene therapy mediated by AAV5 vector can restore the cone function of Cnga3Cldfl5 panchromatic blindness mouse model.
Methods: Cnga3cpfl5 was injected into the subretinal cavity of mice 21 days after birth with AAV5-IRBP/GNAT2-hCNGA3 carrier. More than 80% of the mice with retinal detachment without complications were injected with AAV5-IRBP/GNAT2-hCNGA3 carrier for subsequent experimental observation. After the rats were sacrificed with cervical dislocation under anesthesia, the eyeballs were embedded and frozen sections were made. The expression of retinal optin and CNGA3 proteins was detected by immunofluorescence using the antibodies against optin and CNGA3.
Results: Compared with the non-injected eyes, the function of cone cells was significantly restored after gene therapy. The expression of CNGA3 and opsin was detected in the outer segment of cone cells of Cnga3Cpfl5 mouse retina, and the effective time of treatment lasted at least 5-6 months.
CONCLUSION: Gene therapy was performed at 3 weeks postnatal, and the cone cell function of Cnga 3 CPFL 5 mice was restored to a certain extent. The results of this study may be helpful for gene therapy of human panchromatic blindness caused by CNGA3 mutation.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R774.14

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