【摘要】： 背景 視網(wǎng)膜色素變性(retinitis pigmentosa, RP)是視網(wǎng)膜變性中最常見的一組以進(jìn)行性光感受器細(xì)胞及視網(wǎng)膜色素上皮功能喪失為共同表現(xiàn)的致盲性眼病,其具有高度的遺傳和表型異質(zhì)性。根據(jù)RP遺傳方式的不同,可將其分為常染色體顯性遺傳RP (autosomal dominant RP, adRP)、常染色體隱性遺傳RP(autosomal recessive RP, arRP)和X伴性連鎖遺傳RP (X-lined RP, XLRP),少數(shù)表現(xiàn)為雙基因突變遺傳及線粒體遺傳。當(dāng)因缺乏家族史而無法確定其遺傳方式時(shí),則為散發(fā)型RP (sporadic RR,SRP),大多數(shù)SRP表現(xiàn)為arRP。經(jīng)統(tǒng)計(jì),引起adRP、arRP、XLRP的突變基因分別有20、26、2種。RP是發(fā)達(dá)國(guó)家工作年齡中致盲的最普遍原因,全球RP的發(fā)病率約為1／3500,已超過100萬人受累,但目前尚無有效的預(yù)防和治療措施,而對(duì)RP致病基因的確立則可為探討RP發(fā)病機(jī)制,進(jìn)行遺傳咨詢、產(chǎn)前診斷及基因治療創(chuàng)造條件。研究表明,與人類光感受器功能障礙或變性有關(guān)的基因座約有70多個(gè),目前已發(fā)現(xiàn)53個(gè)基因座與RP有關(guān),因此對(duì)RP致病基因的定位研究仍然是分子遺傳學(xué)研究的熱點(diǎn)。 adRP是最常見的RP遺傳方式,目前在已報(bào)道的53個(gè)基因座中,已確定19個(gè)與adRP有關(guān),其中RHO、PRPF31、RP1、IMPDH1及RDS基因是引起adRP常見的突變基因。RHO與PRPF31基因是我國(guó)漢族RP人群中最常見的突變基因,但在白族人群中尚未見報(bào)道。至今,RHO基因已超過120種不同類型的突變被發(fā)現(xiàn),在這些突變中,90%為單個(gè)堿基置換的點(diǎn)突變,一般突變范圍不超過20個(gè)堿基對(duì)。研究發(fā)現(xiàn),該基因突變存在種族差異,在歐美、日本、中國(guó)其突變所占adRP分別為25%、5.9%、7.7%,南美人群中最常見的RHO基因突變類型是Pro23His,而亞洲,所有已報(bào)道的RHO基因突變均集中在羧基末端(QVSPA), Pro347Lue是該區(qū)域中最多見的突變熱點(diǎn),也是全球性的多發(fā)突變位點(diǎn)。PRPF31基因是僅次于RHO基因座的常見adRP基因,其最先是在一個(gè)英國(guó)adRP大家族通過連鎖分析發(fā)現(xiàn)并將其定位在染色體19q13.4, PRPF31基因突變包括錯(cuò)義突變、缺失、插入及剪接位點(diǎn)的改變,目前已有30個(gè)不同位點(diǎn)的突變被發(fā)現(xiàn),其較多見的突變位點(diǎn)位于外顯子5、外顯子6、外顯子7、外顯子8、外顯子11及內(nèi)含子與外顯子交界處。據(jù)統(tǒng)計(jì),在美國(guó)等西方國(guó)家RP1突變是較常見的,占所有RP的7%,其中以位于外顯子4的錯(cuò)義突變(R677X)多見,在我國(guó),其突變較少見。RDS突變可引起很多類型的視網(wǎng)膜變性,目前已發(fā)現(xiàn)超過70個(gè)RDS基因突變與adRP和常染色體顯性黃斑變性(adMD)有關(guān),其中引起adRP的突變位點(diǎn)僅占5%。RDS突變所致的adRP有地區(qū)差異,在美國(guó)及北歐較多見,在我國(guó)等亞洲國(guó)家較少見,RDS基因突變多位于外顯子1。IMPDH1基因突變多見于外顯子7,目前國(guó)內(nèi)外對(duì)IMPDH1基因的突變報(bào)道尚不多,但其突變所致的adRP發(fā)病早,且嚴(yán)重。 在這些已發(fā)現(xiàn)的RP基因座中大多數(shù)是通過家系連鎖分析的方法定位的,連鎖分析是一種成熟的基因定位克隆技術(shù),90年代初Inglehearn、Greenberg等就利用短串聯(lián)重復(fù)序列(short tandem repeat,STR)遺傳標(biāo)記進(jìn)行連鎖分析,分別在染色體7p、17P上發(fā)現(xiàn)了新的adRP致病基因,隨后也相繼有報(bào)道。該技術(shù)是其以二代或二代以上的家系材料為基礎(chǔ),利用人類基因組中存在的遺傳標(biāo)記進(jìn)行分析,觀察標(biāo)記位點(diǎn)與疾病致病基因位點(diǎn)在家系內(nèi)是否呈共分離,并計(jì)算出遺傳距離及連鎖程度,從而確定致病的候選基因。 遺傳病具有一定的家族遺傳傾向,利用家系對(duì)遺傳性疾病的研究是基因定位的一大特色,也是發(fā)揮我國(guó)人口資源優(yōu)勢(shì)的一大特色。RP致病基因的突變是多種多樣的,特別是在不同地區(qū)、不同民族中,不同基因的突變可導(dǎo)致RP,相同基因的不同突變也可引起RP。白族是我國(guó)西南地區(qū)一個(gè)不同于漢族的少數(shù)民族,其世代遷移少,是相對(duì)封閉的種族,具有良好的單基因疾病遺傳背景,對(duì)其致病基因的定位研究,將豐富我國(guó)RP基因的突變圖譜,為創(chuàng)建我國(guó)RP人群遺傳圖譜及基因序列結(jié)構(gòu)功能信息系統(tǒng)提供更多信息。 研究目的 確定該白族adRP家系的發(fā)病與RHO、PRPF31、RP1、RDS及IMPDH1基因的關(guān)系。 方法 1.基因分型在3號(hào)染色體RHO基因上下游選取相距1cM的2個(gè)短串聯(lián)重復(fù)序列STR:D3S3606與D3S1292第二代遺傳標(biāo)記進(jìn)行多重PCR反應(yīng),STR引物序列5’-端加FAM熒光標(biāo)記,根據(jù)熒光標(biāo)記,采用ABI公司3100測(cè)序儀讀取STR等位基因片段大小,收集數(shù)據(jù)后用Gene Mapper version 3.5軟件包進(jìn)行處理。 2.連鎖分析和單倍型分析利用LINKAGE package 5.1軟件包中的MLINK程序?qū)⒚繉?duì)引物與adRP家系致病基因位點(diǎn)進(jìn)行兩點(diǎn)連鎖分析,根據(jù)下列標(biāo)準(zhǔn)判斷遺傳標(biāo)記與疾病的連鎖程度：LOD值≥+3,肯定連鎖；LOD值≤-2,否定連鎖；-2LOD值+3,則需增加樣本量；家系圖及單倍型分析由Cyrillic2.1軟件繪制。 3.測(cè)序利用Primer premier5.0軟件設(shè)計(jì)引物,PCR產(chǎn)物純化后上樣,利用ABI PRISM(?)3100 Genetic Analyze測(cè)序儀對(duì)RHO基因第1-5外顯子及外顯子與內(nèi)含子的交界處序列及PRPF31、RDS、IMPDH1基因多發(fā)突變位點(diǎn)的進(jìn)行序列分析,測(cè)序結(jié)果與GenBank數(shù)據(jù)庫(kù)中核酸序列進(jìn)行比對(duì)。 4.限制性內(nèi)切酶反應(yīng)全部家系成員的RHO基因第5號(hào)外顯子PCR純化產(chǎn)物加入限制性內(nèi)切酶NlaIVI,消化后取樣于非變性聚丙烯酰胺凝膠(native-PAGE)中進(jìn)行電泳分析,從而從另一角度證實(shí)RHO第5號(hào)外顯子的堿基變異。 5.數(shù)據(jù)統(tǒng)計(jì)實(shí)驗(yàn)對(duì)研究中所發(fā)現(xiàn)的4個(gè)SNP位點(diǎn)：c.45G/A (rs7984)、c.70A/G (rs2269736)、(IVS6-78_IVS6-75del4CACA, rs57960425)及IVS6-31C/T(rs2303557)上的基因型與等位基因頻率進(jìn)行列表統(tǒng)計(jì)。因樣本容量太小且樣本中個(gè)體之間不是相互獨(dú)立的,故未做χ2檢驗(yàn)及等統(tǒng)計(jì)分析。 結(jié)果 1.5代22名成員中13人已確診為視網(wǎng)膜色素變性,其中8名男性5名女性。第Ⅳ代年齡9歲(Ⅳ6),第Ⅴ代年齡6歲(V1),目前均已出現(xiàn)夜盲癥狀,但眼底檢查及電生理檢查尚正常。該家系為常染色體顯性遺傳性視網(wǎng)膜色素變性,家系患者間癥狀相似,即自幼出現(xiàn)夜盲,隨年齡的增長(zhǎng),雙眼視力逐漸減退,晚期多出現(xiàn)后囊性白內(nèi)障,眼底改變表現(xiàn)為：視盤色澤呈蠟黃,視網(wǎng)膜血管變細(xì),視網(wǎng)膜后極部廣泛的骨細(xì)胞樣色素沉著,黃斑光反射存在,視網(wǎng)膜電圖桿錐細(xì)胞振幅減低,甚至熄滅。患病成員中除了視網(wǎng)膜色素變性和白內(nèi)障外,無其他眼科疾病,也無其他異常癥狀。 2.對(duì)RHO基因進(jìn)行兩點(diǎn)連鎖分析,結(jié)果顯示當(dāng)重組率θ=0時(shí),D3S3606、D3S1292存在最大LOD值：3.61、4.55,其支持遺傳標(biāo)記D3S3606、D3S1292與該家系adRP疾病存在連鎖；從單倍型分析中可見,D3S3606與D3S1292之間約的1 cM(里摩)區(qū)域與疾病呈共分離,因此提示該家系的致病基因可能在3號(hào)染色體上D3S3606與D3S1292之間相距1cM的區(qū)域,而該區(qū)域目前發(fā)現(xiàn)的唯一與adRP相關(guān)的致病基因是RHO。 3.RHO基因序列分析結(jié)果發(fā)現(xiàn)所有adRP患者RHO基因第5號(hào)外顯子 347位密碼子的第2堿基處均發(fā)生單個(gè)堿基替換(c.1040CT),該位點(diǎn)呈雜合型,其堿基的變異導(dǎo)致脯氨酸(Pro)變?yōu)榱涟彼?Leu)。此外,家系成員中RHO基因外顯子1非編碼區(qū)存在2個(gè)多態(tài)位點(diǎn)：c.45G/A(rs7984),c.70A/G(rs2269736),統(tǒng)計(jì)發(fā)現(xiàn),61.54%(8／13)患者共表達(dá)AA(rs7984).GG(rs2269736)基因型。而在PRPF31基因中,50%(11／22)家系成員存在PRPF31基因第6內(nèi)含子4個(gè)堿基的缺失(IVS6.78_IVS6.75del4CACA,rs57960425),其中包括7例(53.8%)adRP患者和4例(44.4%)正常個(gè)體；50%(11／22)家系成員存在IVS6-31 C/T(rs2303557)變異,包括8例(61.5%)adRP患者和3例(33.3%)正常個(gè)體。RP1、RDS及IMPDH1基因的多發(fā)位點(diǎn)篩查未發(fā)現(xiàn)任何堿基變異。 4.限制性內(nèi)切酶反應(yīng)顯示,所有家系的RP患者經(jīng)NlaIV酶消化后產(chǎn)生5個(gè)片段：307bp.177bp.130bp.50bp及43bp,而正常個(gè)體只有4個(gè)片段：177bp和130bp.50bp.43bp,因而進(jìn)一步驗(yàn)證了RHO基因第5號(hào)外顯子347位密碼子的第2堿基處發(fā)生了改變,且該位點(diǎn)的突變與疾病呈共分離。 結(jié)論 1.PRPF31.IMPDH1及RDS基因多發(fā)位點(diǎn)的堿基變異與該白族家系的adRP發(fā)病無相關(guān)。 2.RHO基因突變與該白族家系的致病基因存在連鎖關(guān)系,其與adRP疾病呈共分離,所有家系患者均有RHO基因第5號(hào)外顯子的錯(cuò)義突變(p.Pro347Leu),該突變與其致病相關(guān)。 3.RHO基因第1號(hào)外顯子非編碼區(qū)的兩個(gè)SNP位點(diǎn)rs7984(AA). rs2269736(GG)純合基因型的共表達(dá)與p.Pro347Leu突變致病可能存在疾病關(guān)聯(lián)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1

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