中國常染色體顯性視網(wǎng)膜色素變性家系致病基因篩查定位
發(fā)布時間：2018-08-14 17:07
【摘要】：目的： 對收集的42個常染色體顯性遺傳視網(wǎng)膜色素變性(autosomal dominant retinitis pigmentosa, ADRP)家系進行遺傳學病因研究,并對確定基因突變家系的先證者進行臨床表型特點初步總結(jié)�！� 1.在目前已知與ADRP相關(guān)的致病基因中,選擇13個突變頻率較高的基因,通過基因直接測序技術(shù)對42個家系的先證者進行突變篩查； 2.通過多重連接依賴探針擴增(mutiplex ligation-dependent probe amplication,MLPA)反應(yīng)對直接測序未能確定基因突變的家系進行RHO、IMPDH1、RP1和PRPF31基因的巨大缺失突變檢測； 3.在通過上述實驗未確定突變基因的家系中,選擇資料較為全面的3個家系進行全基因組掃描連鎖分析確定致病基因的染色體定位；并在定位區(qū)域內(nèi)選擇候選基因進行測序篩查； 4.對本研究確定基因突變位點的家系進行臨床表型特點的初步總結(jié)； 5.對本研究所確定的新發(fā)突變位點(已知基因)進行初步的突變蛋白功能研究。 方法： 1.臨床研究方法：(1)眼科常規(guī)檢查。包括詢問家族史、病史、最佳矯正視力(best correct visual acuity,BCVA)、眼前節(jié)裂隙燈檢查、直接和間接檢眼鏡檢查及眼底照相等。(2)眼科特殊檢查。包括動態(tài)視野檢查、頻域相干光斷層掃描(optical coherence tomography,OCT)、視網(wǎng)膜電圖(electroretinogram, ERG)檢查等。 2.分子遺傳學研究方法：(1)取所有家系成員外周靜脈血5-8ml,經(jīng)典酚-氯仿法抽提基因組DNA,-20℃冰箱保存?zhèn)溆谩?2)已知候選基因篩查。通過查閱文獻,在所有已知與ADRP相關(guān)的致病基因中,選擇突變頻率較高的13個基因,分別對42個ADRP家系的先證者進行直接基因測序分析。(3) RHO、IMPDH1、RP1和PRPF31基因的巨大突變篩查。通過上述對已知候選基因篩查未能確定突變基因的剩余ADRP家系,對先證者進行針對RHO、IMPDH1、RP1和PRPF31基因的MLPA檢測潛在的巨大基因缺失突變即基因半合子狀態(tài)(hemizygousity)。(4)遺傳連鎖分析。包括①全基因組掃描連鎖分析——選擇Illumina公司illumine HD芯片試劑盒,對通過上述已知候選基因直接篩查測序及MLPA檢測未確定突變的三個ADRP家系進行全基因組掃描連鎖分析。該芯片共包含5913個SNPs位點,原始數(shù)據(jù)經(jīng)Illumina BeadStudio Software v3.1.8讀取基因型,采用MERLIN v1.01軟件進行多點參數(shù)及非參數(shù)連鎖分析；②連鎖區(qū)域內(nèi)候選基因序列分析——根據(jù)連鎖分析的結(jié)果,在所定位的染色體候選區(qū)內(nèi)選取已知與ADRP相關(guān)的致病基因作為候選基因,并采用Sanger末端終止法對候選基因進行直接測序分析。(5)臨床表型特點初步總結(jié)。對確定突變基因的家系先證者的臨床表型特點,包括最佳矯正視力、眼底表現(xiàn)、OCT特征、視野及ERG改變等分別進行初步總結(jié)分析。(6)突變蛋白功能研究。對本研究所確定的其中一個新發(fā)點突變位點(RHO基因,錯義突變L59R)進行突變蛋白功能研究。首先,構(gòu)建突變型RHO基因真核表達載體。在野生型RHO基因真核表達載體RHO-pCMV6-AC-GFP的基礎(chǔ)上,利用定點突變技術(shù),構(gòu)建L59R突變型RHO基因真核表達載體。其次,激光掃描共聚焦顯微鏡觀察蛋白在人神經(jīng)母細胞瘤細胞(SK-N-SH)中的表達和分布。體外培養(yǎng)SK-N-SH細胞,將野生型以及突變型RHO基因真核表達載體RHO-pCMV6-AC-GFP轉(zhuǎn)染至SK-N-SH細胞,利用激光共聚焦顯微鏡分別檢測野生型以及突變型RHO蛋白在細胞內(nèi)外的表達及定位。 結(jié)果： 1.已知候選基因直接篩查測序及MLPA檢測。通過對已知13個ADRP相關(guān)致病基因進行直接篩查測序,在42個ADRP家系中確定了12個家系的致病基因突變位點,其中攜帶PRPF31基因突變的家系有5個(11.9%)、攜帶RHO基因突變的家系有4個(9.5%)、攜帶RP1基因突變的家系有1個(2.4%)、攜帶PRPF3基因突變的家系有1個(2.4%)、攜帶PRPF8基因突變的家系有1個(2.4%)。除此之外,通過對剩余30個ADRP家系進行RHO、IMPDH1、RP1和PRPF31基因的MLPA檢測,我們在其中2個家系(ADRP-SHZ和ADRP-WZZ家系)中發(fā)現(xiàn)其攜帶PRPF31基因的巨大缺失突變,突變率為4.8%(2/42)。 2.連鎖分析。在進行全基因組掃描連鎖分析的3個ADRP家系中,均未發(fā)現(xiàn)明顯突出的連鎖信號。家系A(chǔ)DRP-RLY在遺傳標記物rs2033108和rs2870775之間均取得最大LOD值1.7953,該染色體區(qū)域(染色體17)包含一個已知ADRP候選基因CA4,在該家系中選擇兩位患者對該基因位點的進行擴增后直接雙向測序,結(jié)果并未發(fā)現(xiàn)任何致病性突變。 3.臨床表型特點初步總結(jié)。本研究中確定突變基因的14位先證者初次就診于我院時年齡在24-58之間,患者均以自幼夜盲為主訴,最佳矯正視力在手動～1.0之間。14位患者眼底表現(xiàn)較為相似,主要表現(xiàn)為不同程度的視網(wǎng)膜上骨細胞樣色素沉積及萎縮性病變。OCT檢查示神經(jīng)上皮層、IS/OS層以及視網(wǎng)膜色素上皮層可有不同程度的變薄甚至消失。自動視野檢查可見患者均存在嚴重的向心性視野縮小。ERG檢查可見患者視桿及視錐反應(yīng)波幅顯著降低,部分患者雙眼呈熄滅型。 4.體外成功構(gòu)建突變型RHO基因真核表達載體,激光掃描共聚焦顯微鏡證實野生型RHO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RHO蛋白則明顯堆積于胞漿中,呈大小不等顆粒狀。 結(jié)論： 1.在納入本研究的42個ADRP家系中,通過對已知13個ADRP相關(guān)基因的直接篩查測序,共確定12個家系(28.6%)的基因突變位點,其中PRPF31基因和RHO基因在中國ADRP患者中的突變率較高,分別約占11.9%和9.5%；RPl基因.PRPF3基因和PRPF8基因分別占2.4%。另外,在FSCN2、 PRPH2、IMPDH1、CRX、RPE65、RDH12、GUCA1B及KLHL7基因中,并未發(fā)現(xiàn)任何致病性突變。MLPA檢測共發(fā)現(xiàn)有2個家系攜帶PRPF31基因的巨大缺失突變,突變率約為4.8%。 2.對3個ADRP家系的全基因組掃描分析結(jié)果顯示3個家系與已知ADRP相關(guān)致病基因并未存在連鎖關(guān)系,提示可能有新的致病基因與這3個家系相關(guān)。 3.在本研究中確定突變基因的先證者均表現(xiàn)為典型視網(wǎng)膜色素變性,但病情輕重程度有顯著不同；本研究并未發(fā)現(xiàn)基因型與表型的相關(guān)性。 4.通過對本研究中所確定的RHO基因新發(fā)錯義突變L59R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。
[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.
【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】：2013
【分類號】：R774.13
本文編號：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.
【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】：2013
【分類號】：R774.13
【參考文獻】
相關(guān)期刊論文 前1條
1 張馨方;盛迅倫;;視網(wǎng)膜色素變性的相關(guān)基因研究進展[J];國際眼科雜志;2006年03期
，本文編號：2183536
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