Identification of a novel p.R1443W mutation in RP1 gene associated with retinitis pigmentosa sine pigmento

AIM:To screen mutations in the retinitis pigmentosa 1(RP1) gene and the rhodopsin(RHO) gene in Chinese patients with retinitis pigmentosa sine pigmento(RPSP)and describe the genotype-phenotype relationship of the mutations.·METHODS:Twenty affected,unrelated Chinese individuals with RPSP(4 autosomal dominant RPSP,12autosomal recessive RPSP and 4 unknown inheritance pattern) were recruited between 2009 and 2012.The clinical features were determined by complete ophthalmologic examinations.Polymerase chain reaction(PCR) and direct DNA sequencing were used to screen the entire coding region and splice junctions of the RP1gene and the RHO gene.The cosegregation analysis and population frequency studies were performed for patients with identified mutations.·RESULTS:Five variants in the RP1 gene and one in the RHO gene were detected in 20 probands.Four missense changes(rs444772,rs446227,rs414352,rs441800) and one non-coding variant(rs56340615) were common SNPs and none of them showed a significant relationship with RPSP.A missense mutation p.R1443W was identified in the RP1 gene in three affected individuals from a family with autosomal dominant RPSP and was found to cosegregate with the phenotype in this family,suggestive of pathogenic.In addition,population frequency analysis showed the p.R1443W mutation was absent in 300 healthy controls.·CONCLUSION:The identification of p.R1443W mutationcosegregating in a family with autosomal dominant RPSP highlights an atypical phenotype of the RP1 gene mutation,while RHO gene is not associated with the pathogenesis of RPSP in this study.To our knowledge,this is the fist mutation identified to associate with RPSP.

110例视网膜色素变性患者RP1基因突变的检测分析

背景视网膜色素变性（RP）是一组常见的单基因遗传性、致盲性眼底病变，目前尚无有效的治疗方法。目的研究RP1基因在宁夏地区RP患者中的突变频率及特征，进一步探讨其在RP发病机制中的潜在作用。方法收集宁夏回族自治区110例RP患者，其中包含35例常染色体显性遗传性RP（ADRP）患者和75例散发患者为RP患者组，同时收集100例健康成年人为正常对照组，2组均采集外周静脉血3～5ml，应用聚合酶链反应（PCR）和直接测序法进行RP1基因全编码区及邻近剪切位点内含子区域序列突变的检测。运用多因素Logistic回归方法和基于网络的评分软件PolyPhen、Sorting Intolerantfrom Tolerant（SIFT）对研究结果进行分析。结果RP患者组和正常对照组RPl基因共检测出11个变异位点，P．Lysll52Lys和C．＊247A〉C为新发现的突变。RP患者组第3内含子C．788—92T〉C的突变频率与正常对照组比较差异有统计学意义（χ2=9．12，P〈0．01）；3’-侧翼序列区c．＊247A〉C的突变频率高于正常对照组，差异有统计学意义（χ2=12．77，P〈0．01），且与RP发生呈显著正相关（r=1．11，P〈0．05），其余位点突变均证实为RPl基因的多态性。RP患者组P．Gln1725Gin的突变率显著高于正常对照组（χ2=42．09，P〈0．01），但其与RP的发生无关（r=1．74，P〉0．05）。1例散发患者发现了P．Lysll52Lys的突变。在83例RP患者中发现有P．Arg872His、P．Alal670Thr和P．Serl69lPro突变的同时出现，PolyPhen分析显示，三者均为保护性突变，评分分别为1．11、0．74、0．22；SIFT分析显示，这3个突变均对RP表现为保护作用，评分分别为0．07、0．60、0．22；携带这3个突变的RP患者夜盲出现的平均年龄为30．54岁，27例未携带这3个突变的RP患者夜盲出现的平均年龄为21．06岁；携带这3个突变的RP患者平均最佳矫正视力为0．50±0．38，未携带者的为0．40±0．33，差异有统计学意义（t=2．11，P〈0．05）。结论在宁夏回族自治区RP患者中，RPl基因的致病突变率低于中国其他地区的人群。P．Arg872His、P．Alal670Thr和P．Serl691Pro突变的协同出现可能对RP起到一定的保护作用，同时也可能降低了RPl基因致病突变的发生率。

SCREENING FOR MUTATIONS IN A NOVEL RETINAL - SPECIFIC GENE AMONG CHINESE PATIENTS WITH RETINITIS PIGMENTOSA

Objective. To identify and evaluate mutations in the RPl gene among Chinese patients with retinitis pigmen-tosa (RP).Methods. Leukocyte DNA of 92 RP patients were collected in Hong Kong. Sequence changes of the entire coding region of the RP1 gene were examined using PCR, conformation sensitive gel electrophoresis and DNA sequencing.Results. In total, 1 nonsense mutation and 1 nonsense variant as well as 10 missense alterations were identified in the RP1 gene, among which, Arg677Ter was found in 1 RP patient and another nonsense variant, Argl933Ter, was identified in 3 normal individuals and 1 patient with Stargardt' s disease, suggesting its nonpathogenicity. Arg677Ter is expected to lead to large disruptions of the encoded protein.Conclusions. The nonpathogenicity of Argl933Ter indicates that the C - terminal 224 residues of RPl protein may be not critical for RP1. The most C - terminal truncation previously reported was due to Tyr1053 (1 -bp del) and occurred in RP patients. Thus RP can be caused by reduction in the level of the region of RPl protein after codon 1052 but before 1933. To ascertain such a proposition, genotypes of more RP patients may reveal more RP causative mutations and more sequence alterations different than those of other ethnic groups.

胰岛素样生长因子结合蛋白相关蛋白1基因在儿童急性白血病中的表达及临床意义

目的:探讨胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor-binding protein related protein 1,IGFBP-rP1)基因在儿童急性白血病中的表达及临床意义。方法:应用实时荧光定量PCR(real-time fluorescence quantitative-PCR,RFQ-PCR)法检测168例(次)急性白血病患儿骨髓细胞中IGFBP-rP1 mRNA的表达水平,并与同期30例非白血病患儿作对照。根据儿童急性白血病的预后因素,分析IGFBP-rP1 mRNA的表达与患者临床预后的关系。结果:初诊组急性白血病患儿的IGFBP-rP1 mRNA表达水平明显高于非白血病患儿(P<0.01)。初诊组急性髓细胞白血病(acute myelocytic leukemia,AML)患儿的IGFBP-rP1 mRNA表达水平高于急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)患儿(P=0.013)。完全缓解(complete remission,CR)组AML患儿的IGFBP-rP1 mRNA表达最低,接近非白血病患儿组;复发组AML患儿的IGFBP-rP1 mRNA表达水平再度上升,明显高于CR时的表达水平。而ALL患儿的IGFBP-rP1 mRNA表达水平在初诊组、CR组及复发组间的差异无统计学意义。结论:IGFBP-rP1基因可能参与儿童AML的发生和发展,并有望成为新的治疗靶点。

喉癌组织中IGFBP-rP1基因的甲基化及表达

目的:探讨喉癌中胰岛素样生长因子结合蛋白-相关蛋白1(IGFBP-rP1)基因启动子区甲基化与蛋白表达之间的关系。方法:采用甲基化特异性PCR方法和免疫组织化学的方法分别检测45例喉癌组织及18例癌旁组织中IGFBP-rP1基因的甲基化状态和蛋白表达情况。结果:喉癌组织IGFBP-rP1启动子区甲基化率为33.3%(15/45),相应癌旁组织甲基化率为5.6%(1/18),喉癌组织中IGFBP-rP1基因甲基化率明显高于癌旁组织(P<0.05)。喉癌组织中IGFBP-rP1蛋白表达显著低于癌旁正常组织(P<0.05),且与其启动子区甲基化状态呈负相关。结论:IGFBP-rP1基因启动子区甲基化导致基因沉默可能是喉癌发生的机制之一。

RbAp46基因激活IGFBP-rPl基因在K562细胞中的表达

目的 探讨视网膜母细胞瘤相关蛋白46（RbAp46）基因对白血病细胞的作用机制。方法 将全长RbAp46基因的cDNA转染人白血病细胞系K562细胞及人脑膜胶质瘤细胞系SHG44细胞，建立稳定过度表达RbAp46基因的细胞株，观察细胞的生物学行为的同时，通过RT—PCR方法寻找表达差异的相关基因。结果 过度表达RbAp46基因的K562细胞及SHG44细胞生长受抑。表现在K562／RbAp46细胞与K562／CMV细胞于生长第4天分别为（90．00±8．40）×10^4和（119．58±9．87）×10^4，SHG44／RbAp46细胞与SHG44／CMV细胞于生长第5天分别为（89．13±4．88）×10^4和（149．42±10．83）×10^4。生长第7天时，K562／RbAp46细胞和K562／CMV细胞集落数分别为131．67±15．57和250．33±26．31（P〈0．01），SHG44／RbAp46细胞和SHG44／CMV细胞集落数分别为50．78±6．77和206．67±37．18（P〈0．01）。K562／RbA046细胞与K562／CMV细胞S期细胞分别占（48．88±4.35）％和（62．78±4．78）％（P〈0．01），G0／G1期细胞分别占（29．10±4．14）％和（22．40±2．43）％（P〈0．05），而SHG44／RbAp46细胞与SHG44／CMV细胞G0／G1期细胞分别占65．6％和48．8％，S期细胞分别占22．6％和38．4％。过度表达RbAp46基因的K562细胞出现胰岛素样生长因子结合蛋白相关蛋白1（IGFBP—rP1）基因的诱导性表达。结论 在K562细胞中可能存在RbAp46和IGFBP-rP1基因之间的调控通路。