一中国白族常染色体显性遗传视网膜色素变性家系基因定位研究
发布时间：2018-08-12 14:58
【摘要】： 背景 视网膜色素变性(retinitis pigmentosa, RP)是视网膜变性中最常见的一组以进行性光感受器细胞及视网膜色素上皮功能丧失为共同表现的致盲性眼病,其具有高度的遗传和表型异质性。根据RP遗传方式的不同,可将其分为常染色体显性遗传RP (autosomal dominant RP, adRP)、常染色体隐性遗传RP(autosomal recessive RP, arRP)和X伴性连锁遗传RP (X-lined RP, XLRP),少数表现为双基因突变遗传及线粒体遗传。当因缺乏家族史而无法确定其遗传方式时,则为散发型RP (sporadic RR,SRP),大多数SRP表现为arRP。经统计,引起adRP、arRP、XLRP的突变基因分别有20、26、2种。RP是发达国家工作年龄中致盲的最普遍原因,全球RP的发病率约为1／3500,已超过100万人受累,但目前尚无有效的预防和治疗措施,而对RP致病基因的确立则可为探讨RP发病机制,进行遗传咨询、产前诊断及基因治疗创造条件。研究表明,与人类光感受器功能障碍或变性有关的基因座约有70多个,目前已发现53个基因座与RP有关,因此对RP致病基因的定位研究仍然是分子遗传学研究的热点。 adRP是最常见的RP遗传方式,目前在已报道的53个基因座中,已确定19个与adRP有关,其中RHO、PRPF31、RP1、IMPDH1及RDS基因是引起adRP常见的突变基因。RHO与PRPF31基因是我国汉族RP人群中最常见的突变基因,但在白族人群中尚未见报道。至今,RHO基因已超过120种不同类型的突变被发现,在这些突变中,90%为单个碱基置换的点突变,一般突变范围不超过20个碱基对。研究发现,该基因突变存在种族差异,在欧美、日本、中国其突变所占adRP分别为25%、5.9%、7.7%,南美人群中最常见的RHO基因突变类型是Pro23His,而亚洲,所有已报道的RHO基因突变均集中在羧基末端(QVSPA), Pro347Lue是该区域中最多见的突变热点,也是全球性的多发突变位点。PRPF31基因是仅次于RHO基因座的常见adRP基因,其最先是在一个英国adRP大家族通过连锁分析发现并将其定位在染色体19q13.4, PRPF31基因突变包括错义突变、缺失、插入及剪接位点的改变,目前已有30个不同位点的突变被发现,其较多见的突变位点位于外显子5、外显子6、外显子7、外显子8、外显子11及内含子与外显子交界处。据统计,在美国等西方国家RP1突变是较常见的,占所有RP的7%,其中以位于外显子4的错义突变(R677X)多见,在我国,其突变较少见。RDS突变可引起很多类型的视网膜变性,目前已发现超过70个RDS基因突变与adRP和常染色体显性黄斑变性(adMD)有关,其中引起adRP的突变位点仅占5%。RDS突变所致的adRP有地区差异,在美国及北欧较多见,在我国等亚洲国家较少见,RDS基因突变多位于外显子1。IMPDH1基因突变多见于外显子7,目前国内外对IMPDH1基因的突变报道尚不多,但其突变所致的adRP发病早,且严重。 在这些已发现的RP基因座中大多数是通过家系连锁分析的方法定位的,连锁分析是一种成熟的基因定位克隆技术,90年代初Inglehearn、Greenberg等就利用短串联重复序列(short tandem repeat,STR)遗传标记进行连锁分析,分别在染色体7p、17P上发现了新的adRP致病基因,随后也相继有报道。该技术是其以二代或二代以上的家系材料为基础,利用人类基因组中存在的遗传标记进行分析,观察标记位点与疾病致病基因位点在家系内是否呈共分离,并计算出遗传距离及连锁程度,从而确定致病的候选基因。 遗传病具有一定的家族遗传倾向,利用家系对遗传性疾病的研究是基因定位的一大特色,也是发挥我国人口资源优势的一大特色。RP致病基因的突变是多种多样的,特别是在不同地区、不同民族中,不同基因的突变可导致RP,相同基因的不同突变也可引起RP。白族是我国西南地区一个不同于汉族的少数民族,其世代迁移少,是相对封闭的种族,具有良好的单基因疾病遗传背景,对其致病基因的定位研究,将丰富我国RP基因的突变图谱,为创建我国RP人群遗传图谱及基因序列结构功能信息系统提供更多信息。 研究目的 确定该白族adRP家系的发病与RHO、PRPF31、RP1、RDS及IMPDH1基因的关系。 方法 1.基因分型在3号染色体RHO基因上下游选取相距1cM的2个短串联重复序列STR:D3S3606与D3S1292第二代遗传标记进行多重PCR反应,STR引物序列5’-端加FAM荧光标记,根据荧光标记,采用ABI公司3100测序仪读取STR等位基因片段大小,收集数据后用Gene Mapper version 3.5软件包进行处理。 2.连锁分析和单倍型分析利用LINKAGE package 5.1软件包中的MLINK程序将每对引物与adRP家系致病基因位点进行两点连锁分析,根据下列标准判断遗传标记与疾病的连锁程度：LOD值≥+3,肯定连锁；LOD值≤-2,否定连锁；-2LOD值+3,则需增加样本量；家系图及单倍型分析由Cyrillic2.1软件绘制。 3.测序利用Primer premier5.0软件设计引物,PCR产物纯化后上样,利用ABI PRISM(?)3100 Genetic Analyze测序仪对RHO基因第1-5外显子及外显子与内含子的交界处序列及PRPF31、RDS、IMPDH1基因多发突变位点的进行序列分析,测序结果与GenBank数据库中核酸序列进行比对。 4.限制性内切酶反应全部家系成员的RHO基因第5号外显子PCR纯化产物加入限制性内切酶NlaIVI,消化后取样于非变性聚丙烯酰胺凝胶(native-PAGE)中进行电泳分析,从而从另一角度证实RHO第5号外显子的碱基变异。 5.数据统计实验对研究中所发现的4个SNP位点：c.45G/A (rs7984)、c.70A/G (rs2269736)、(IVS6-78_IVS6-75del4CACA, rs57960425)及IVS6-31C/T(rs2303557)上的基因型与等位基因频率进行列表统计。因样本容量太小且样本中个体之间不是相互独立的,故未做χ2检验及等统计分析。 结果 1.5代22名成员中13人已确诊为视网膜色素变性,其中8名男性5名女性。第Ⅳ代年龄9岁(Ⅳ6),第Ⅴ代年龄6岁(V1),目前均已出现夜盲症状,但眼底检查及电生理检查尚正常。该家系为常染色体显性遗传性视网膜色素变性,家系患者间症状相似,即自幼出现夜盲,随年龄的增长,双眼视力逐渐减退,晚期多出现后囊性白内障,眼底改变表现为：视盘色泽呈蜡黄,视网膜血管变细,视网膜后极部广泛的骨细胞样色素沉着,黄斑光反射存在,视网膜电图杆锥细胞振幅减低,甚至熄灭。患病成员中除了视网膜色素变性和白内障外,无其他眼科疾病,也无其他异常症状。 2.对RHO基因进行两点连锁分析,结果显示当重组率θ=0时,D3S3606、D3S1292存在最大LOD值：3.61、4.55,其支持遗传标记D3S3606、D3S1292与该家系adRP疾病存在连锁；从单倍型分析中可见,D3S3606与D3S1292之间约的1 cM(里摩)区域与疾病呈共分离,因此提示该家系的致病基因可能在3号染色体上D3S3606与D3S1292之间相距1cM的区域,而该区域目前发现的唯一与adRP相关的致病基因是RHO。 3.RHO基因序列分析结果发现所有adRP患者RHO基因第5号外显子 347位密码子的第2碱基处均发生单个碱基替换(c.1040CT),该位点呈杂合型,其碱基的变异导致脯氨酸(Pro)变为亮氨酸(Leu)。此外,家系成员中RHO基因外显子1非编码区存在2个多态位点：c.45G/A(rs7984),c.70A/G(rs2269736),统计发现,61.54%(8／13)患者共表达AA(rs7984).GG(rs2269736)基因型。而在PRPF31基因中,50%(11／22)家系成员存在PRPF31基因第6内含子4个碱基的缺失(IVS6.78_IVS6.75del4CACA,rs57960425),其中包括7例(53.8%)adRP患者和4例(44.4%)正常个体；50%(11／22)家系成员存在IVS6-31 C/T(rs2303557)变异,包括8例(61.5%)adRP患者和3例(33.3%)正常个体。RP1、RDS及IMPDH1基因的多发位点筛查未发现任何碱基变异。 4.限制性内切酶反应显示,所有家系的RP患者经NlaIV酶消化后产生5个片段：307bp.177bp.130bp.50bp及43bp,而正常个体只有4个片段：177bp和130bp.50bp.43bp,因而进一步验证了RHO基因第5号外显子347位密码子的第2碱基处发生了改变,且该位点的突变与疾病呈共分离。 结论 1.PRPF31.IMPDH1及RDS基因多发位点的碱基变异与该白族家系的adRP发病无相关。 2.RHO基因突变与该白族家系的致病基因存在连锁关系,其与adRP疾病呈共分离,所有家系患者均有RHO基因第5号外显子的错义突变(p.Pro347Leu),该突变与其致病相关。 3.RHO基因第1号外显子非编码区的两个SNP位点rs7984(AA). rs2269736(GG)纯合基因型的共表达与p.Pro347Leu突变致病可能存在疾病关联性。
[Abstract]:background
Retinitis pigmentosa (RP) is the most common group of blinding ophthalmopathy with progressive photoreceptor cell and retinal pigment epithelial dysfunction, which has high genetic and phenotypic heterogeneity. P (autosomal dominant RP, adRP), autosomal recessive RP (arRP) and X-linked RP (XLRP), a few of which were inherited by two-gene mutation and mitochondrial inheritance. According to statistics, there are 20,26,2 mutation genes causing adRP, arRP and XLRP respectively. RP is the most common cause of blindness in working age in developed countries. The incidence of global RP is about 1/3500, and more than 1 million people are affected. However, there is no effective preventive and therapeutic measures to establish the pathogenic gene of RP. Genetic counseling, prenatal diagnosis and gene therapy create conditions. Studies have shown that there are more than 70 loci associated with photoreceptor dysfunction or degeneration in humans, and 53 loci have been found to be related to RP.
AdRP is the most common form of RP inheritance. Of the 53 reported loci, 19 have been identified to be associated with adRP. RHO, PRPF31, RP1, IMPDH1 and RDS are the most common mutations causing adRP. More than 120 different types of mutations have been found. 90% of these mutations are point mutations with a single base substitution and the general mutation range is no more than 20 base pairs. Pro23His is the type, whereas in Asia, all reported RHO mutations are concentrated in the carboxyl terminal (QVSPA). Pro347Lue is the most common mutation hotspot in the region and a global multiple mutation site. The mutations of PRPF31 gene include missense mutation, deletion, insertion and splicing sites. At present, 30 different mutations have been found. The most common mutation sites are exon 5, exon 6, exon 7, exon 8, exon 11 and the junction of intron and exon. The mutation of RP1 is common in Western countries, accounting for 7% of all RPs, especially in exon 4 missense mutation (R677X), which is rare in China. RDS mutation can cause many types of retinal degeneration. Up to now, more than 70 RDS gene mutations have been found to be associated with adRP and autosomal dominant macular degeneration (adMD). The mutation sites of ADRP were only 5%. RDS mutation caused by ADRP was more common in the United States and Northern Europe, and less common in China and other Asian countries. The mutation of RDS gene was more common in exon 1. IMPDH1 gene was more common in exon 7. Mutations of IMPDH1 gene were seldom reported at home and abroad, but the mutation caused by ADRP was early in onset. It's serious.
In the early 1990s, Inglehearn and Greenberg used short tandem repeat (STR) genetic markers for linkage analysis, which were found on chromosomes 7p and 17P respectively. New adRP pathogenic genes have been reported in succession. This technique is based on the second generation or more family materials, using genetic markers existing in the human genome to analyze, observe whether marker sites and disease-causing gene loci are co-segregated within the family, and calculate the genetic distance and linkage degree, so as to be sure. Candidate genes for pathogenicity.
Genetic diseases have a certain family hereditary tendency. The study of genetic diseases by families is a major feature of gene mapping, and also a major feature of giving full play to the advantages of population resources in China. The same mutation can also cause RP. Bai nationality is a minority nationality in southwest China, which is different from Han nationality. It is a relatively closed race and has a good genetic background of single gene disease. The mapping study of its pathogenic gene will enrich the mutation map of RP gene in China and create the genetic map and sequence of RP population in China. The structural function information system provides more information.
research objective
The relationship between the adRP family and the RHO, PRPF31, RP1, RDS and IMPDH1 genes were determined.
Method
1. Two short tandem repeats STR:D3S3606 and D3S1292 were selected from the upstream and downstream of RHO gene on chromosome 3 for multiplex PCR. The 5'-end of STR primer sequence was labeled with FAM fluorescent marker. According to the fluorescent marker, the STR allele fragment size was read by ABI 3100 sequencer, and the data were collected by Gen. E Mapper version 3.5 software package for processing.
2. Linkage analysis and haplotype analysis were performed by using the MLINK program in LINKAGE package 5.1. Each pair of primers was linked to the pathogenic gene loci of adRP families, and the degree of linkage between genetic markers and diseases was determined according to the following criteria: LOD value (>+3), affirmative linkage; LOD value (< 2), negative linkage; and - 2LOD value + 3, additional samples were needed. This volume, family diagram and haplotype analysis were drawn by Cyrillic2.1 software.
3. Primer premier 5.0 software was used to design primers, PCR products were purified and sampled, and ABI PRISM (?) 3100 Genetic Analyser was used to sequence the RHO gene exon 1-5 and the exon-intron junction as well as the PRPF31, RDS, IMPDH1 gene mutant sites. The sequencing results were compared with those of GenBank database. Sequence alignment.
4. The PCR purified product of exon 5 of RHO gene in all family members of restriction endonuclease reaction was added to restriction endonuclease NlaIVI. After digestion, samples were taken from non-denatured polyacrylamide gel (native-PAGE) for electrophoresis analysis. The base variation of exon 5 of RHO gene was confirmed from another point of view.
5. The genotype and allele frequencies of the four SNP loci found in the study were tabulated by data statistics experiment: c.45G/A (rs7984), c.70A/G (rs2269736), (IVS6-78_IVS6-75del4CACA, rs57960425) and IVS6-31C/T (rs2303557). And statistical analysis.
Result
13 of the 22 members of the 1.5 generation have been diagnosed with retinitis pigmentosa, including 8 males and 5 females. The fourth generation is 9 years old (IV6) and the fifth generation is 6 years old (V1). Nocturnal blindness has occurred, but fundus examination and electrophysiological examination are still normal. That is, night blindness occurs at an early age, with the growth of age, the visual acuity of both eyes gradually declines, and in the late stage, posterior capsular cataract often occurs. Fundus changes are as follows: the color of the optic disc is waxy yellow, the retinal blood vessels become thinner, the posterior pole of the retina is extensive osteocyte-like pigmentation, macular light reflex exists, the cone cell amplitude of the electroretinogram rod is reduced, or even extinguished. In addition to retinitis pigmentosa and cataract, there were no other ophthalmic diseases and other abnormal symptoms.
2. Two-point linkage analysis of RHO gene showed that D3S3606 and D3S1292 had the maximum LOD value of 3.61,4.55 when the recombination rate was 0, which supported genetic markers D3S3606 and D3S1292 to be linked to adRP disease in the family. From the haplotype analysis, it was found that about 1 cM (Rimo) region between D3S3606 and D3S1292 was separated from the disease. The pathogenic gene of this family may be in the region 1 cm apart between D3S3606 and D3S1292 on chromosome 3, and the only pathogenic gene associated with adRP found in this region is RHO.
Sequence analysis of 3.RHO gene revealed that exon fifth of RHO gene was found in all adRP patients.
A single base substitution (c.1040CT) occurred at the 2nd base of codon 347, which was heterozygous and resulted in proline (Pro) becoming leucine (Leu). In addition, there were two polymorphic loci in exon 1 of RHO gene: c.45G/A (rs7984) and c.70A/G (rs2269736). Statistically, 61.54% (8/13) of the family members had a total of patients. AA (rs7984). GG (rs2269736) genotype was expressed. In PRPF31 gene, 50% (11/22) of the family members had deletions of 4 bases in intron 6 of PRPF31 gene (IVS6.78_IVS6.75del4CA, rs57960425), including 7 cases (53.8%) of adRP and 4 cases (44.4%) of normal individuals; 50% (11/22) of the family members had IVS6-31 C/T (rs2303557) mutation. There were 8 cases (61.5%) of adRP and 3 cases (33.3%) of normal individuals. No base mutation was found in multiple site screening for RP1, RDS and IMPDH1 genes.
4. Restriction endonuclease reaction showed that five fragments were produced by NlaIV digestion in all families: 307 bp. 177 bp. 130 bp. 50 BP and 43 bp, while only four fragments were found in normal individuals: 177 BP and 130 bp. 50 bp. 43 bp. This further confirmed that the second base of codon 347 of exon 5 of the RHO gene had been changed and the site was protruded. The disease is separated from the disease.
conclusion
1. The base mutations of PRPF31. IMPDH1 and RDS gene polymorphisms were not associated with the onset of adRP in this Bai family.
2. The mutation of RHO gene was associated with the pathogenic gene of the Bai family. It was co-segregated with adRP disease. All families had missense mutation in exon 5 of RHO gene (p. Pro347Leu), which was associated with the pathogenesis of ADRP.
3. The co-expression of two homozygous genotypes of rs7984 (AA). rs2269736 (GG) in the non-coding region of exon 1 of RHO gene may be associated with the pathogenesis of p.Pro347Leu mutation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1
[Abstract]:background
Retinitis pigmentosa (RP) is the most common group of blinding ophthalmopathy with progressive photoreceptor cell and retinal pigment epithelial dysfunction, which has high genetic and phenotypic heterogeneity. P (autosomal dominant RP, adRP), autosomal recessive RP (arRP) and X-linked RP (XLRP), a few of which were inherited by two-gene mutation and mitochondrial inheritance. According to statistics, there are 20,26,2 mutation genes causing adRP, arRP and XLRP respectively. RP is the most common cause of blindness in working age in developed countries. The incidence of global RP is about 1/3500, and more than 1 million people are affected. However, there is no effective preventive and therapeutic measures to establish the pathogenic gene of RP. Genetic counseling, prenatal diagnosis and gene therapy create conditions. Studies have shown that there are more than 70 loci associated with photoreceptor dysfunction or degeneration in humans, and 53 loci have been found to be related to RP.
AdRP is the most common form of RP inheritance. Of the 53 reported loci, 19 have been identified to be associated with adRP. RHO, PRPF31, RP1, IMPDH1 and RDS are the most common mutations causing adRP. More than 120 different types of mutations have been found. 90% of these mutations are point mutations with a single base substitution and the general mutation range is no more than 20 base pairs. Pro23His is the type, whereas in Asia, all reported RHO mutations are concentrated in the carboxyl terminal (QVSPA). Pro347Lue is the most common mutation hotspot in the region and a global multiple mutation site. The mutations of PRPF31 gene include missense mutation, deletion, insertion and splicing sites. At present, 30 different mutations have been found. The most common mutation sites are exon 5, exon 6, exon 7, exon 8, exon 11 and the junction of intron and exon. The mutation of RP1 is common in Western countries, accounting for 7% of all RPs, especially in exon 4 missense mutation (R677X), which is rare in China. RDS mutation can cause many types of retinal degeneration. Up to now, more than 70 RDS gene mutations have been found to be associated with adRP and autosomal dominant macular degeneration (adMD). The mutation sites of ADRP were only 5%. RDS mutation caused by ADRP was more common in the United States and Northern Europe, and less common in China and other Asian countries. The mutation of RDS gene was more common in exon 1. IMPDH1 gene was more common in exon 7. Mutations of IMPDH1 gene were seldom reported at home and abroad, but the mutation caused by ADRP was early in onset. It's serious.
In the early 1990s, Inglehearn and Greenberg used short tandem repeat (STR) genetic markers for linkage analysis, which were found on chromosomes 7p and 17P respectively. New adRP pathogenic genes have been reported in succession. This technique is based on the second generation or more family materials, using genetic markers existing in the human genome to analyze, observe whether marker sites and disease-causing gene loci are co-segregated within the family, and calculate the genetic distance and linkage degree, so as to be sure. Candidate genes for pathogenicity.
Genetic diseases have a certain family hereditary tendency. The study of genetic diseases by families is a major feature of gene mapping, and also a major feature of giving full play to the advantages of population resources in China. The same mutation can also cause RP. Bai nationality is a minority nationality in southwest China, which is different from Han nationality. It is a relatively closed race and has a good genetic background of single gene disease. The mapping study of its pathogenic gene will enrich the mutation map of RP gene in China and create the genetic map and sequence of RP population in China. The structural function information system provides more information.
research objective
The relationship between the adRP family and the RHO, PRPF31, RP1, RDS and IMPDH1 genes were determined.
Method
1. Two short tandem repeats STR:D3S3606 and D3S1292 were selected from the upstream and downstream of RHO gene on chromosome 3 for multiplex PCR. The 5'-end of STR primer sequence was labeled with FAM fluorescent marker. According to the fluorescent marker, the STR allele fragment size was read by ABI 3100 sequencer, and the data were collected by Gen. E Mapper version 3.5 software package for processing.
2. Linkage analysis and haplotype analysis were performed by using the MLINK program in LINKAGE package 5.1. Each pair of primers was linked to the pathogenic gene loci of adRP families, and the degree of linkage between genetic markers and diseases was determined according to the following criteria: LOD value (>+3), affirmative linkage; LOD value (< 2), negative linkage; and - 2LOD value + 3, additional samples were needed. This volume, family diagram and haplotype analysis were drawn by Cyrillic2.1 software.
3. Primer premier 5.0 software was used to design primers, PCR products were purified and sampled, and ABI PRISM (?) 3100 Genetic Analyser was used to sequence the RHO gene exon 1-5 and the exon-intron junction as well as the PRPF31, RDS, IMPDH1 gene mutant sites. The sequencing results were compared with those of GenBank database. Sequence alignment.
4. The PCR purified product of exon 5 of RHO gene in all family members of restriction endonuclease reaction was added to restriction endonuclease NlaIVI. After digestion, samples were taken from non-denatured polyacrylamide gel (native-PAGE) for electrophoresis analysis. The base variation of exon 5 of RHO gene was confirmed from another point of view.
5. The genotype and allele frequencies of the four SNP loci found in the study were tabulated by data statistics experiment: c.45G/A (rs7984), c.70A/G (rs2269736), (IVS6-78_IVS6-75del4CACA, rs57960425) and IVS6-31C/T (rs2303557). And statistical analysis.
Result
13 of the 22 members of the 1.5 generation have been diagnosed with retinitis pigmentosa, including 8 males and 5 females. The fourth generation is 9 years old (IV6) and the fifth generation is 6 years old (V1). Nocturnal blindness has occurred, but fundus examination and electrophysiological examination are still normal. That is, night blindness occurs at an early age, with the growth of age, the visual acuity of both eyes gradually declines, and in the late stage, posterior capsular cataract often occurs. Fundus changes are as follows: the color of the optic disc is waxy yellow, the retinal blood vessels become thinner, the posterior pole of the retina is extensive osteocyte-like pigmentation, macular light reflex exists, the cone cell amplitude of the electroretinogram rod is reduced, or even extinguished. In addition to retinitis pigmentosa and cataract, there were no other ophthalmic diseases and other abnormal symptoms.
2. Two-point linkage analysis of RHO gene showed that D3S3606 and D3S1292 had the maximum LOD value of 3.61,4.55 when the recombination rate was 0, which supported genetic markers D3S3606 and D3S1292 to be linked to adRP disease in the family. From the haplotype analysis, it was found that about 1 cM (Rimo) region between D3S3606 and D3S1292 was separated from the disease. The pathogenic gene of this family may be in the region 1 cm apart between D3S3606 and D3S1292 on chromosome 3, and the only pathogenic gene associated with adRP found in this region is RHO.
Sequence analysis of 3.RHO gene revealed that exon fifth of RHO gene was found in all adRP patients.
A single base substitution (c.1040CT) occurred at the 2nd base of codon 347, which was heterozygous and resulted in proline (Pro) becoming leucine (Leu). In addition, there were two polymorphic loci in exon 1 of RHO gene: c.45G/A (rs7984) and c.70A/G (rs2269736). Statistically, 61.54% (8/13) of the family members had a total of patients. AA (rs7984). GG (rs2269736) genotype was expressed. In PRPF31 gene, 50% (11/22) of the family members had deletions of 4 bases in intron 6 of PRPF31 gene (IVS6.78_IVS6.75del4CA, rs57960425), including 7 cases (53.8%) of adRP and 4 cases (44.4%) of normal individuals; 50% (11/22) of the family members had IVS6-31 C/T (rs2303557) mutation. There were 8 cases (61.5%) of adRP and 3 cases (33.3%) of normal individuals. No base mutation was found in multiple site screening for RP1, RDS and IMPDH1 genes.
4. Restriction endonuclease reaction showed that five fragments were produced by NlaIV digestion in all families: 307 bp. 177 bp. 130 bp. 50 BP and 43 bp, while only four fragments were found in normal individuals: 177 BP and 130 bp. 50 bp. 43 bp. This further confirmed that the second base of codon 347 of exon 5 of the RHO gene had been changed and the site was protruded. The disease is separated from the disease.
conclusion
1. The base mutations of PRPF31. IMPDH1 and RDS gene polymorphisms were not associated with the onset of adRP in this Bai family.
2. The mutation of RHO gene was associated with the pathogenic gene of the Bai family. It was co-segregated with adRP disease. All families had missense mutation in exon 5 of RHO gene (p. Pro347Leu), which was associated with the pathogenesis of ADRP.
3. The co-expression of two homozygous genotypes of rs7984 (AA). rs2269736 (GG) in the non-coding region of exon 1 of RHO gene may be associated with the pathogenesis of p.Pro347Leu mutation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1
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