視錐細胞功能異常眼病的致病基因研究和全色盲小鼠模型的基因治療
發(fā)布時間：2018-09-09 19:04
【摘要】：目的：Alstrom綜合征(Alstrom syndrome, AS)是一種少見的常染色體隱性遺傳病,臨床表現(xiàn)時視錐視桿細胞營養(yǎng)不良和多器官功能障礙。目前唯一已知的致病基因ALMS1基因突變可引起Alstrom綜合征。本研究的目的是鑒定Alstrom綜合征患者的致病突變并評價該綜合征的臨床特點。 方法：收集患者病史,進行眼部和與該疾病相關的全身檢查,并采集患者及其父母靜脈血,提取基因組DNA。對致病基因ALMS1的外顯子、外顯子和內(nèi)含子交界區(qū)序列進行PCR (polymerase chain reaction, PCR1擴增,目的產(chǎn)物切膠純化后直接測序。與基因庫中ALMS1的轉(zhuǎn)錄本(NM015120.4)進行比對,排除單核苷酸多態(tài)(single nucleotide polymorphism, SNP)并確定致病突變。 結果：共有來自5個家系的7名患者診斷為Alstrom綜合征,并且這些家系都是非近親家系。這些患者均有視錐視桿細胞營養(yǎng)不良的臨床表現(xiàn),視力差,畏光和眼球震顫。另外患者還伴有感音性耳聾、肥胖、胰島素抵抗、2型糖尿病、肝腎功能障礙、高胰島素血癥、甲狀腺功能減退、智力障礙、黑棘皮癥和脊柱側(cè)凸等其他臨床癥狀�；驒z測結果發(fā)現(xiàn)：患者1的兩個新移碼突變p.N3150Kfs2X和p.V3154Xfs;患者2的新移碼突變p.N3672Ifs11X和曾經(jīng)報道過的錯義突變p.R3703X;患者3的新錯義突變和移碼突變p.S2479X和p.R3611Efs7X;患者4和5的未曾報道的純合錯義突變p.S695X;患者6和7的新移碼突變p.H688HfsX和p.Q3147Qfs2X。在100個無關的健康對照人群中沒有發(fā)現(xiàn)這些突變。共分離分析顯示父母分別是這些突變等位基因的雜合攜帶者。 結論：本研究中7名患者全部都具有Alstrom綜合征典型的眼部異常和多系統(tǒng)異常。這些ALMS1的突變導致翻譯錯誤的截短的蛋白不能發(fā)揮正常功能,是該綜合征發(fā)病的遺傳基礎。 目的：研究全色盲患者的臨床表現(xiàn)的特征并確定致病基因突變。 方法：分別來自于10個家系的15名全色盲患者進行分析。收集患者病史,進行眼部相關檢查包括BCVA、色覺、裂隙燈檢查、眼底檢查、視網(wǎng)膜電流圖檢查、視野檢查和黃斑部的SD-OCT檢查。采集患者及其父母靜脈血,提取基因組DNA。對致病基因CNGA3、CNGB3、GNAT2、PDE6C和PDE6H的外顯子、外顯子和內(nèi)含子交界區(qū)進行PCR擴增,目的產(chǎn)物切膠純化后直接測序。運用Blat tool工具與基因庫中標準序列進行比對,排除SNP并確定致病突變,并在家系里進行共分離分析驗證突變。突變結果在1000genomes進行比對排除SNP。 結果：患者均表現(xiàn)為畏光、眼球震顫和辨色力完全喪失或殘存部分辨色能力�；颊叩淖罴殉C正視力在0.05～0.2。視錐細胞ERG反應未記錄到波形或振幅顯著降低,SD-OCT示IS/OS層不同程度消失和黃斑區(qū)視網(wǎng)膜厚度變薄。共有8個家系13名患者均發(fā)現(xiàn)CNGA3突變。這些突變包括7個新的錯義突變和3個新的缺失突變和4個曾經(jīng)報導過的錯義突變。另外2名患者沒有檢測到5個已知致病基因的任何突變。 結論：CNGA3是中國全色盲患者最常見的致病基因。本研究發(fā)現(xiàn)10個新的CNGA3突變。發(fā)現(xiàn)全色盲患者的遺傳學特點對于遺傳咨詢和將來的基因治療都很關鍵。本研究是目前關于中國全色盲患者的首次遺傳學報道。 目的：對上一部分研究所確定的CNGA3新發(fā)純合錯義突變進行初步的體外突變蛋白功能研究。 方法：針對CNGA3的純合錯義突變D211E的蛋白功能研究,構建突變型CNGA3基因真核表達載體。在野生型CNGA3基因真核表達載體CNGA3-pCMV6的基礎上,利用定點誘變技術,構建D211E突變型CNGA3基因真核表達載體。體外培養(yǎng)HEK293細胞,將野生型以及突變型CNGA3基因真核表達載體CNGA3-pCMV6轉(zhuǎn)染至HEK293細胞,經(jīng)免疫熒光染色,熒光顯微鏡觀察比較野生型以及突變型CNGA3蛋白在人胚腎細胞(HEK293)中的表達和分布。 結果：成功構建突變型CNGA3基因真核表達載體,體外轉(zhuǎn)染至HEK293細胞后,野生型與突變型蛋白分布差異很大。熒光顯微鏡證實野生型CNGA3蛋白主要分布于細胞膜,在細胞胞漿內(nèi)僅有少量呈點狀、散在分布,而突變型CNGA3蛋白則明顯聚集在一起堆積于胞漿中。 結論：通過對本研究中所確定的CNGA3基因新發(fā)錯義突變D211E進行突變蛋白功能研究,提示該突變會明顯影響蛋白在細胞中的分布,失去功能的蛋白質(zhì)是引起發(fā)病的分子機制。 目的：探討AAV5載體介導的基因治療能否恢復全色盲小鼠模型Cnga3Cldfl5的視錐細胞功能。 方法：對出生后21天的Cnga3cpfl5進行視網(wǎng)膜下腔注射AAV5-IRBP/GNAT2-hCNGA3載體,注射后視網(wǎng)膜脫離達80%以上并且沒有并發(fā)癥的小鼠用于后續(xù)的實驗觀察。小鼠只接受注射單側(cè)眼,另一眼用作對照觀察。注射載體5-6月時記錄視網(wǎng)膜電流圖。小鼠麻醉頸椎脫臼處死后,將眼球包埋進行冰凍切片。用視蛋白抗體和CNGA3抗體作為一抗,免疫熒光檢測視網(wǎng)膜的視蛋白和CNGA3蛋白是否表達。 結果：和未注射眼相比較,經(jīng)過基因治療視錐細胞的功能得到了明顯的恢復。Cnga3Cpfl5小鼠視網(wǎng)膜視錐細胞外節(jié)檢測到了CNGA3和視蛋白的表達,并且治療有效的時間至少維持了5-6個月。 結論：本研究是在生后3周進行基因治療的,而且使自然發(fā)生的全色盲小鼠Cnga3cpfl5小鼠視錐細胞功能得到了一定程度的恢復。該研究結果對于人類CNGA3突變引起的全色盲的基因治療有一定的啟示作用。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R774.14
本文編號：2233294
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R774.14
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