中国常染色体显性视网膜色素变性家系致病基因筛查定位
发布时间：2018-08-14 17:07
【摘要】：目的： 对收集的42个常染色体显性遗传视网膜色素变性(autosomal dominant retinitis pigmentosa, ADRP)家系进行遗传学病因研究,并对确定基因突变家系的先证者进行临床表型特点初步总结。’ 1.在目前已知与ADRP相关的致病基因中,选择13个突变频率较高的基因,通过基因直接测序技术对42个家系的先证者进行突变筛查； 2.通过多重连接依赖探针扩增(mutiplex ligation-dependent probe amplication,MLPA)反应对直接测序未能确定基因突变的家系进行RHO、IMPDH1、RP1和PRPF31基因的巨大缺失突变检测； 3.在通过上述实验未确定突变基因的家系中,选择资料较为全面的3个家系进行全基因组扫描连锁分析确定致病基因的染色体定位；并在定位区域内选择候选基因进行测序筛查； 4.对本研究确定基因突变位点的家系进行临床表型特点的初步总结； 5.对本研究所确定的新发突变位点(已知基因)进行初步的突变蛋白功能研究。 方法： 1.临床研究方法：(1)眼科常规检查。包括询问家族史、病史、最佳矫正视力(best correct visual acuity,BCVA)、眼前节裂隙灯检查、直接和间接检眼镜检查及眼底照相等。(2)眼科特殊检查。包括动态视野检查、频域相干光断层扫描(optical coherence tomography,OCT)、视网膜电图(electroretinogram, ERG)检查等。 2.分子遗传学研究方法：(1)取所有家系成员外周静脉血5-8ml,经典酚-氯仿法抽提基因组DNA,-20℃冰箱保存备用。(2)已知候选基因筛查。通过查阅文献,在所有已知与ADRP相关的致病基因中,选择突变频率较高的13个基因,分别对42个ADRP家系的先证者进行直接基因测序分析。(3) RHO、IMPDH1、RP1和PRPF31基因的巨大突变筛查。通过上述对已知候选基因筛查未能确定突变基因的剩余ADRP家系,对先证者进行针对RHO、IMPDH1、RP1和PRPF31基因的MLPA检测潜在的巨大基因缺失突变即基因半合子状态(hemizygousity)。(4)遗传连锁分析。包括①全基因组扫描连锁分析——选择Illumina公司illumine HD芯片试剂盒,对通过上述已知候选基因直接筛查测序及MLPA检测未确定突变的三个ADRP家系进行全基因组扫描连锁分析。该芯片共包含5913个SNPs位点,原始数据经Illumina BeadStudio Software v3.1.8读取基因型,采用MERLIN v1.01软件进行多点参数及非参数连锁分析；②连锁区域内候选基因序列分析——根据连锁分析的结果,在所定位的染色体候选区内选取已知与ADRP相关的致病基因作为候选基因,并采用Sanger末端终止法对候选基因进行直接测序分析。(5)临床表型特点初步总结。对确定突变基因的家系先证者的临床表型特点,包括最佳矫正视力、眼底表现、OCT特征、视野及ERG改变等分别进行初步总结分析。(6)突变蛋白功能研究。对本研究所确定的其中一个新发点突变位点(RHO基因,错义突变L59R)进行突变蛋白功能研究。首先,构建突变型RHO基因真核表达载体。在野生型RHO基因真核表达载体RHO-pCMV6-AC-GFP的基础上,利用定点突变技术,构建L59R突变型RHO基因真核表达载体。其次,激光扫描共聚焦显微镜观察蛋白在人神经母细胞瘤细胞(SK-N-SH)中的表达和分布。体外培养SK-N-SH细胞,将野生型以及突变型RHO基因真核表达载体RHO-pCMV6-AC-GFP转染至SK-N-SH细胞,利用激光共聚焦显微镜分别检测野生型以及突变型RHO蛋白在细胞内外的表达及定位。 结果： 1.已知候选基因直接筛查测序及MLPA检测。通过对已知13个ADRP相关致病基因进行直接筛查测序,在42个ADRP家系中确定了12个家系的致病基因突变位点,其中携带PRPF31基因突变的家系有5个(11.9%)、携带RHO基因突变的家系有4个(9.5%)、携带RP1基因突变的家系有1个(2.4%)、携带PRPF3基因突变的家系有1个(2.4%)、携带PRPF8基因突变的家系有1个(2.4%)。除此之外,通过对剩余30个ADRP家系进行RHO、IMPDH1、RP1和PRPF31基因的MLPA检测,我们在其中2个家系(ADRP-SHZ和ADRP-WZZ家系)中发现其携带PRPF31基因的巨大缺失突变,突变率为4.8%(2/42)。 2.连锁分析。在进行全基因组扫描连锁分析的3个ADRP家系中,均未发现明显突出的连锁信号。家系ADRP-RLY在遗传标记物rs2033108和rs2870775之间均取得最大LOD值1.7953,该染色体区域(染色体17)包含一个已知ADRP候选基因CA4,在该家系中选择两位患者对该基因位点的进行扩增后直接双向测序,结果并未发现任何致病性突变。 3.临床表型特点初步总结。本研究中确定突变基因的14位先证者初次就诊于我院时年龄在24-58之间,患者均以自幼夜盲为主诉,最佳矫正视力在手动～1.0之间。14位患者眼底表现较为相似,主要表现为不同程度的视网膜上骨细胞样色素沉积及萎缩性病变。OCT检查示神经上皮层、IS/OS层以及视网膜色素上皮层可有不同程度的变薄甚至消失。自动视野检查可见患者均存在严重的向心性视野缩小。ERG检查可见患者视杆及视锥反应波幅显著降低,部分患者双眼呈熄灭型。 4.体外成功构建突变型RHO基因真核表达载体,激光扫描共聚焦显微镜证实野生型RHO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RHO蛋白则明显堆积于胞浆中,呈大小不等颗粒状。 结论： 1.在纳入本研究的42个ADRP家系中,通过对已知13个ADRP相关基因的直接筛查测序,共确定12个家系(28.6%)的基因突变位点,其中PRPF31基因和RHO基因在中国ADRP患者中的突变率较高,分别约占11.9%和9.5%；RPl基因.PRPF3基因和PRPF8基因分别占2.4%。另外,在FSCN2、 PRPH2、IMPDH1、CRX、RPE65、RDH12、GUCA1B及KLHL7基因中,并未发现任何致病性突变。MLPA检测共发现有2个家系携带PRPF31基因的巨大缺失突变,突变率约为4.8%。 2.对3个ADRP家系的全基因组扫描分析结果显示3个家系与已知ADRP相关致病基因并未存在连锁关系,提示可能有新的致病基因与这3个家系相关。 3.在本研究中确定突变基因的先证者均表现为典型视网膜色素变性,但病情轻重程度有显著不同；本研究并未发现基因型与表型的相关性。 4.通过对本研究中所确定的RHO基因新发错义突变L59R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。
[Abstract]:Objective:
The genetic etiology of 42 autosomal dominant retinitis pigmentosa (ADRP) pedigrees was studied and the clinical phenotypic characteristics of the probands who identified the gene mutation pedigrees were preliminarily summarized.
1. Among the known pathogenic genes related to ADRP, 13 genes with high mutation frequency were selected and the probands from 42 families were screened by direct gene sequencing.
2. Mutiplex ligation-dependent probe amplication (MLPA) was used to detect large deletion mutations in RHO, IMPDH1, RP1 and PRPF31 genes in families with undetermined gene mutations.
3. In the families with undetermined mutant genes, three families with more comprehensive data were selected for genome-wide scan linkage analysis to determine the chromosome location of the pathogenic genes, and candidate genes were selected for sequencing and screening in the localization region.
4. to summarize the clinical phenotypes of families with gene mutation sites identified in this study.
5. the preliminary mutation protein function of the new mutation site (known gene) identified in this study was studied.
Method:
1. Clinical research methods: (1) Routine ophthalmic examination, including family history, medical history, best correct visual acuity (BCVA), slit lamp examination of anterior segment, direct and indirect ophthalmoscopy and fundus photography, etc. (2) Special ophthalmic examination, including dynamic visual field examination, frequency domain coherence tomography, etc. Aphy, OCT), electroretinogram (ERG) examination and so on.
2. Molecular genetic research methods: (1) Genomic DNA was extracted from peripheral venous blood of all family members by classical phenol-chloroform method and stored in refrigerator at - 20 C for reserve. (2) Screening of known candidate genes. The proband was analyzed by direct gene sequencing. (3) Large mutation screening of RHO, IMPDH1, RP1 and PRPF31 genes. The MLPA of RHO, IMPDH1, RP1 and PRPF31 genes was used to detect potential large gene deletion mutations, i.e. gene hemizygote, in the remaining ADRP families whose mutation genes could not be determined by the above screening of known candidate genes. (4) Genetic linkage analysis. Including: (1) Whole genome scan linkage analysis - Illumine HD chip kit was selected for genome scan linkage analysis in three ADRP families with undetermined mutations detected by MLPA and direct screening of the known candidate genes. S locus, the original data was read by Illumina Bead Studio Software v3.1.8 genotype, using MERLIN v1.01 software for multi-point parameter and non-parameter linkage analysis; 2 Linkage region candidate gene sequence analysis - based on the results of linkage analysis, in the located chromosome candidate region to select known pathogenic genes associated with ADRP for production. For candidate genes, Sanger terminal termination method was used to direct sequencing analysis of candidate genes. (5) Preliminary summary of clinical phenotypic characteristics. The mutant protein function of a new point mutation site (RHO gene, missense mutation L59R) was studied. Firstly, the mutant RHO gene eukaryotic expression vector was constructed. Based on the wild RHO gene eukaryotic expression vector RHO-pCMV6-AC-GFP, the L59R mutant RHO gene was constructed by site-directed mutagenesis. Eukaryotic expression vectors. Secondly, the expression and distribution of the proteins in human neuroblastoma cells (SK-N-SH) were observed by laser scanning confocal microscopy. SK-N-SH cells were cultured in vitro, and wild-type and mutant RHO gene eukaryotic expression vectors RHO-pCMV6-AC-GFP were transfected into SK-N-SH cells, and the wild-type was detected by laser scanning confocal microscopy. And the expression and location of mutant RHO protein in and out of cells.
Result:
1. Direct screening and sequencing of known candidate genes and MLPA detection. Through direct screening and sequencing of 13 known ADRP-related pathogenic genes, 12 mutation sites of pathogenic genes were identified in 42 ADRP families, including 5 (11.9%) families with PRPF31 gene mutation, 4 (9.5%) families with RHO gene mutation and 4 (9.5%) families with RP1 gene mutation. There were 1 (2.4%) families with PRPF3 gene mutation, 1 (2.4%) families with PRPF8 gene mutation and 1 (2.4%) families with PRPF8 gene mutation. In addition, we found PRPF31 gene in 2 of the remaining 30 ADRP families (ADRP-SHZ and ADRP-WZZ families) by MLPA detection of RHO, IMPDH1, RP1 and PRPF31 genes. The mutation rate was 4.8% (2/42).
2. Linkage analysis. None of the three ADRP families performed genome-wide scan linkage analysis showed significant linkage signals. ADRP-RLY obtained a maximum LOD value of 1.7953 between the genetic markers rs2033108 and rs2870775. The chromosome region (chromosome 17) contained a known candidate gene CA4 for ADRP, and two of the families were selected. The gene was amplified and sequenced directly in two directions. No pathogenic mutation was found.
3. Preliminary summary of clinical phenotypic characteristics. 14 probands with mutant genes were found to be between 24 and 58 years old at the time of their first visit to our hospital. All patients complained of night blindness from childhood. The best corrected visual acuity was between 1.0 and 1.0. The fundus performance of 14 patients was comparatively similar, mainly showing different degrees of osteocyte-like pigmentation in the retina. OCT showed that the neuroepithelial layer, IS/OS layer and retinal pigment epithelial layer were thinned or even disappeared in varying degrees. Automated visual field examination showed severe centripetal visual field reduction. ERG showed that the amplitude of rod and cone response was significantly reduced, and some patients had extinct eyes.
4. Eukaryotic expression vector of mutant RHO gene was successfully constructed in vitro. Laser scanning confocal microscopy confirmed that wild-type RHO protein was mainly distributed in the cell membrane, only a few of them were dotted and scattered in the cytoplasm, while mutant RHO protein was obviously accumulated in the cytoplasm, showing granular shape.
Conclusion:
1. In 42 ADRP families included in this study, 12 families (28.6%) were identified by direct screening and sequencing of 13 known ADRP-related genes. The mutation rates of PRPF31 and RHO genes were higher in Chinese ADRP patients, accounting for 11.9% and 9.5% respectively, and that of RPl gene, PRPF3 gene and PRPF8 gene accounted for 2.4% respectively. In addition, no pathogenic mutation was found in FSCN2, PRPH2, IMPDH1, CRX, RPE65, RDH12, GUCA1B and KLHL7 genes. MLPA detection revealed that two families carried large deletion mutations of PRPF31 gene, the mutation rate was about 4.8%.
2. The whole genome scan analysis of three ADRP families showed that there was no linkage between the three families and the known ADRP-related pathogenic genes, suggesting that there might be a new pathogenic gene associated with the three families.
3. In this study, all the probands who identified the mutant gene showed typical retinitis pigmentosa, but the severity of the disease was significantly different.
4. The function of a new missense mutation L59R in the RHO gene identified in this study was studied, suggesting that the mutation could significantly affect the protein distribution in cells.
【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】：2013
【分类号】：R774.13
本文编号：2183536
[Abstract]:Objective:
The genetic etiology of 42 autosomal dominant retinitis pigmentosa (ADRP) pedigrees was studied and the clinical phenotypic characteristics of the probands who identified the gene mutation pedigrees were preliminarily summarized.
1. Among the known pathogenic genes related to ADRP, 13 genes with high mutation frequency were selected and the probands from 42 families were screened by direct gene sequencing.
2. Mutiplex ligation-dependent probe amplication (MLPA) was used to detect large deletion mutations in RHO, IMPDH1, RP1 and PRPF31 genes in families with undetermined gene mutations.
3. In the families with undetermined mutant genes, three families with more comprehensive data were selected for genome-wide scan linkage analysis to determine the chromosome location of the pathogenic genes, and candidate genes were selected for sequencing and screening in the localization region.
4. to summarize the clinical phenotypes of families with gene mutation sites identified in this study.
5. the preliminary mutation protein function of the new mutation site (known gene) identified in this study was studied.
Method:
1. Clinical research methods: (1) Routine ophthalmic examination, including family history, medical history, best correct visual acuity (BCVA), slit lamp examination of anterior segment, direct and indirect ophthalmoscopy and fundus photography, etc. (2) Special ophthalmic examination, including dynamic visual field examination, frequency domain coherence tomography, etc. Aphy, OCT), electroretinogram (ERG) examination and so on.
2. Molecular genetic research methods: (1) Genomic DNA was extracted from peripheral venous blood of all family members by classical phenol-chloroform method and stored in refrigerator at - 20 C for reserve. (2) Screening of known candidate genes. The proband was analyzed by direct gene sequencing. (3) Large mutation screening of RHO, IMPDH1, RP1 and PRPF31 genes. The MLPA of RHO, IMPDH1, RP1 and PRPF31 genes was used to detect potential large gene deletion mutations, i.e. gene hemizygote, in the remaining ADRP families whose mutation genes could not be determined by the above screening of known candidate genes. (4) Genetic linkage analysis. Including: (1) Whole genome scan linkage analysis - Illumine HD chip kit was selected for genome scan linkage analysis in three ADRP families with undetermined mutations detected by MLPA and direct screening of the known candidate genes. S locus, the original data was read by Illumina Bead Studio Software v3.1.8 genotype, using MERLIN v1.01 software for multi-point parameter and non-parameter linkage analysis; 2 Linkage region candidate gene sequence analysis - based on the results of linkage analysis, in the located chromosome candidate region to select known pathogenic genes associated with ADRP for production. For candidate genes, Sanger terminal termination method was used to direct sequencing analysis of candidate genes. (5) Preliminary summary of clinical phenotypic characteristics. The mutant protein function of a new point mutation site (RHO gene, missense mutation L59R) was studied. Firstly, the mutant RHO gene eukaryotic expression vector was constructed. Based on the wild RHO gene eukaryotic expression vector RHO-pCMV6-AC-GFP, the L59R mutant RHO gene was constructed by site-directed mutagenesis. Eukaryotic expression vectors. Secondly, the expression and distribution of the proteins in human neuroblastoma cells (SK-N-SH) were observed by laser scanning confocal microscopy. SK-N-SH cells were cultured in vitro, and wild-type and mutant RHO gene eukaryotic expression vectors RHO-pCMV6-AC-GFP were transfected into SK-N-SH cells, and the wild-type was detected by laser scanning confocal microscopy. And the expression and location of mutant RHO protein in and out of cells.
Result:
1. Direct screening and sequencing of known candidate genes and MLPA detection. Through direct screening and sequencing of 13 known ADRP-related pathogenic genes, 12 mutation sites of pathogenic genes were identified in 42 ADRP families, including 5 (11.9%) families with PRPF31 gene mutation, 4 (9.5%) families with RHO gene mutation and 4 (9.5%) families with RP1 gene mutation. There were 1 (2.4%) families with PRPF3 gene mutation, 1 (2.4%) families with PRPF8 gene mutation and 1 (2.4%) families with PRPF8 gene mutation. In addition, we found PRPF31 gene in 2 of the remaining 30 ADRP families (ADRP-SHZ and ADRP-WZZ families) by MLPA detection of RHO, IMPDH1, RP1 and PRPF31 genes. The mutation rate was 4.8% (2/42).
2. Linkage analysis. None of the three ADRP families performed genome-wide scan linkage analysis showed significant linkage signals. ADRP-RLY obtained a maximum LOD value of 1.7953 between the genetic markers rs2033108 and rs2870775. The chromosome region (chromosome 17) contained a known candidate gene CA4 for ADRP, and two of the families were selected. The gene was amplified and sequenced directly in two directions. No pathogenic mutation was found.
3. Preliminary summary of clinical phenotypic characteristics. 14 probands with mutant genes were found to be between 24 and 58 years old at the time of their first visit to our hospital. All patients complained of night blindness from childhood. The best corrected visual acuity was between 1.0 and 1.0. The fundus performance of 14 patients was comparatively similar, mainly showing different degrees of osteocyte-like pigmentation in the retina. OCT showed that the neuroepithelial layer, IS/OS layer and retinal pigment epithelial layer were thinned or even disappeared in varying degrees. Automated visual field examination showed severe centripetal visual field reduction. ERG showed that the amplitude of rod and cone response was significantly reduced, and some patients had extinct eyes.
4. Eukaryotic expression vector of mutant RHO gene was successfully constructed in vitro. Laser scanning confocal microscopy confirmed that wild-type RHO protein was mainly distributed in the cell membrane, only a few of them were dotted and scattered in the cytoplasm, while mutant RHO protein was obviously accumulated in the cytoplasm, showing granular shape.
Conclusion:
1. In 42 ADRP families included in this study, 12 families (28.6%) were identified by direct screening and sequencing of 13 known ADRP-related genes. The mutation rates of PRPF31 and RHO genes were higher in Chinese ADRP patients, accounting for 11.9% and 9.5% respectively, and that of RPl gene, PRPF3 gene and PRPF8 gene accounted for 2.4% respectively. In addition, no pathogenic mutation was found in FSCN2, PRPH2, IMPDH1, CRX, RPE65, RDH12, GUCA1B and KLHL7 genes. MLPA detection revealed that two families carried large deletion mutations of PRPF31 gene, the mutation rate was about 4.8%.
2. The whole genome scan analysis of three ADRP families showed that there was no linkage between the three families and the known ADRP-related pathogenic genes, suggesting that there might be a new pathogenic gene associated with the three families.
3. In this study, all the probands who identified the mutant gene showed typical retinitis pigmentosa, but the severity of the disease was significantly different.
4. The function of a new missense mutation L59R in the RHO gene identified in this study was studied, suggesting that the mutation could significantly affect the protein distribution in cells.
【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】：2013
【分类号】：R774.13
【参考文献】
相关期刊论文 前1条
1 张馨方;盛迅伦;;视网膜色素变性的相关基因研究进展[J];国际眼科杂志;2006年03期
，本文编号：2183536
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