眼皮肤白化病Ⅱ型相关的P基因突变与DNA多态性

眼皮肤白化病Ⅱ型(OCA2)是白化病中最常见的类型,呈常染色体隐性遗传。P基因为其致病基因,定位于15q11.1－q12,由24个外显子和23个内含子构成。P基因编码838个氨基酸残基构成的110 KDa跨膜蛋白,该蛋白含12个跨膜区,其确切功能尚未完全清楚。迄今至少已报道P基因内60种导致OCA2的病理性突变和43种多态性变异。病理突变主要为错义突变、无义突变、移码突变和剪切位点突变,多数位于肽链的C末端,但并不象OCA1的TYR基因突变那样多成簇出现。P基因多态性变异中的大部分位于外显子,这增加了对致病性突变定义的难度,其中一些导致氨基酸替换的多态性变异可能与正常人色素沉着的表型变     

眼皮肤白化病分型及珊基因新突变的鉴定

目的了解我国眼皮肤白化病（oculocutaneous albinism,OCA）的分型和相关基因突变类型,探讨新突变可能的分子致病机制。方法应用PCR方法扩增TYR基因,经DNA序列测定检出突变,采用错配引物PCR进行新突变的群体筛查,结合生物信息学方法探讨一种新突变的致病性和可能的分子致病机制。结果10名患者中有5人存在2个突变TYR等位基因,共计8种突变类型,其中c．71G〉A（C24Y）和c．841G〉T（E281X）是OCA1A致病性新突变;C24极可能参与二硫键形成,C24Y将导致酪氨酸酶肽链内此二硫键消失,进而引起蛋白空间构象变化和功能异常而致病。结论从基因水平初步了解了我国OCA1所占的比例,探讨了TYR基因C24Y的致病性并初步阐明了其致病的分子机制。本结果丰富了人类TYR基因突变类型,为我国OCA分型诊断、产前基因诊断和遗传咨询等积累了有价值的数据资料。     

OCA2基因的研究进展

OCA2基因为哺乳动物重要的色素基因,参与黑色素合成反应,同时也是人眼睛颜色的主要调控基因。其编码一个110 kD大小的跨膜蛋白,该蛋白质定位于黑素体膜。研究表明, OCA2主要通过影响黑素体成熟、调节黑素体pH值以及参与黑色素合成途径第一步这3个方面影响黑色素合成。相邻基因HERC2的rs12913832增强子位点通过影响OCA2表达水平,实现对人眼睛颜色的调控。同时, OCA2基因是Ⅱ型眼皮肤白化病(oculocutaneousalbinism typeⅡ, OCA2)的致病基因,与普拉德-威利综合征(Prader-Willi syndrome, PWS)、天使综合征(Angelman syndrome, AS)和黑色素瘤之间也存在间接关联,这些患者通常表现出皮肤或头发低色素沉着。本文从分子生物学、细胞生物学和遗传学的角度对OCA2基因进行概述,以期为进一步探究OCA2的分子机制以及相关疾病的诊疗提供思路和见解。     

眼－皮肤型白化病及其遗传特点

眼－皮肤型白化病及其遗传特点曹熙芳（湖北医学院附属第一医院卫生学校武昌４３００６０）白化病是一种由遗传因素造成黑色素形成障碍的疾病。此病有眼一皮肤型和眼型２类。最常见的是全身皮肤、毛发和眼睛都缺乏色素的白化病，叫眼一皮肤型白化病，本文主要介绍论这种白...     

眼皮肤白化病患者酪氨酸酶基因突变的研究

目的：对临床诊断为眼皮肤白化病（OCA）患者的酪氨酸酶（TYR）基因进行突变筛查,了解我国大陆OCA患者TYR基因突变类型,探讨基因突变对人TYR蛋白结构和功能的影响。方法：应用PCR技术,扩增患者及其父母的TYR基因外显子、外显子-内含子交界区及启动子区;以DNA序列测定技术,进行突变筛查与鉴定;利用生物信息学方法,对突变引起蛋白结构和功能的改变进行预测与分析。结果：在15名患者的30个TYR等位基因内,查明11种突变;其中错义突变5种（W400L、R299H、E294K、R77Q和K142M）,无义突变3种（R116X、R278X和G295X）,插入突变2种（929insC和232insGGG）,剪切位点突变1种（IVS1-3 C〉G）;对4个突变W400L、R299H、929insC、232insGGG的生物信息学分析显示,突变的致病性与蛋白结构和功能的改变相关。结论：W400L占本研究所检出全部OCA1突变等位基因的30．0％（9／30）,可能为中国大陆人群中较常见的TYR基因突变类型;应用生物信息学分析方法对TYR基因突变的致病性做出一些合理可能的解释是可行的。     

大肠杆菌严谨型RNA聚合酶的筛选及体内转录活性测定

利用选择性培养基筛选大肠杆菌自然突变菌株,经噬菌体P1转导和蛋白质互补试验,发现一株突变体(LCH001)的突变基因发生在编码RNA聚合酶β′亚基的rpoC基因上,经DNA序列分析,发现突变位点发生在第3406个碱基上,由G变成了T,导致编码的氨基酸由甘氨酸(GGT)变成半胱氨酸(TGT)。体内转录试验表明该突变RNA聚合酶转录严谨型启动子控制基因的活性显著降低,其β-半乳糖苷酶的活性是野生型菌株的18%,而转录非严谨型启动子控制基因的活性显著提高,其β-半乳糖苷酶的活性约是野生型菌株的5倍。研究结果对探讨RNA聚合酶结构与功能的关系以及RNA聚合酶在细菌严谨反应过程中的作用具有重要意义。     

人类基因组中巢式基因对的系统分析

基因组上一个基因的所有或大部分外显子位于另一基因内含子和UTR中被称为巢式基因(Nested gene)。巢式基因对(Nested gene pair)是由主基因(Host gene)和巢式基因组成的基因对。文章针对人类基因组上的顺式巢式基因对(简称为巢式基因对)进行全基因组鉴定,并通过比较巢式基因对在人和小鼠基因组上的保守性分布,研究巢式基因对的进化方式。保守性分析表明基因转座、序列原位突变和主基因转录起始/终止位点突变是巢式基因对进化的主要原因,其中主基因转录起始/终止位点突变是巢式基因对独特的一种进化方式。Gene On-tology分析揭示大部分巢式基因与主基因的产物在功能上没有相关性。     

蛋白质组学技术筛选和鉴定白化黑线仓鼠皮肤白化相关差异蛋白质

目的比较黑线仓鼠及其白化突变系背部皮肤蛋白表达的差异,寻找差异蛋白质,从蛋白质水平探讨白化病的发生机制。方法应用双向凝胶电泳技术分离出差异蛋白质,用质谱法分析其结构与组成,通过蛋白质数据库确定差异蛋白的功能。结果从64个表达差异蛋白斑点中发现33个显著差异的蛋白点,其中又有14个差异点匹配到了有意义的蛋白质。14个差异点共鉴定出11个差异蛋白质,这些差异蛋白质按功能可分为4类:(1)糖代谢相关蛋白;(2)运输蛋白;(3)细胞骨架蛋白;(4)其他蛋白。结论黑线仓鼠与其白化突变系背部皮肤蛋白表达存在明显差异,其中一些蛋白与白化病发生相关,并可能成为白化病致病机理研究的分子标志物和药物治疗靶向位点。     

Elongin的转录延伸调控及其与VHL肿瘤抑制蛋白的关系

一种新的转录延伸因子Elongin能催化哺乳动物细胞中RNA pclⅡ的转录延伸速度。El-ogin复合体是由分子量分别为110、18、和15KD的A、B、C三种亚基组成的异源三聚体。VHL蛋白对Elongin的转录延伸作用有负调控作用,VHL蛋白由VHL基因(Von Hippel-Lindau tumor suppressor gene)所编码。VHL基因的突变会引起VHL型肿瘤,一种遗传性癌症综合征。VHL蛋白能与Elongin的B,C亚基紧密结合,从而抑制Elongin复合体的转录延伸活力,这可能是VHL型肿瘤抑制的基础。这些发现表明,VHL蛋白在Elongin的转录调控中起着一定的作用。     

人U_1和U_2 snRNA基因5''''端区域中SV_(40)T抗原结合位点的作用

利用寡聚核苷酸指导的基因定位突变技术,从人U_1和U_2 snRNA基因5'端删去或取代能与SV_(40)T抗原结合的位点,构成基因的缺失和取代突变体。在HeLa细胞核抽提物体外转录系统中,U_1和U_2缺失及取代突变基因与野生型基因有相同的转录水平,而且RNA的合成都是从帽子位点的上游开始的。这意味着基因这一区域的突变不改变其体外转录性质。在人的HeLa和293细胞及蛙卵母细胞中,U_1和U_2缺失突变基因不被转录,而取代突变基因的转录却又恢复到野生型基因水平。这表明人的U_1和U_2基因在这三种细胞内的转录与SV_(40)T抗原结合位点的核苷酸排列顺序无关,而由于结合位点的缺失所造成的空间距离上的变化才是影响转录的主要因素。     

Oculocutaneous albinism type 4 is one of the most common types of albinism in Japan

Oculocutaneous albinism (OCA) is a complex genetic disease with great clinical heterogeneity. Four different types of OCA have been reported to date (OCA1, OCA2, OCA3, and OCA4). MATP was recently reported in a single Turkish OCA patient as the fourth pathological gene, but no other patients with OCA4 have been reported. Here, we report the mutational profile of OCA4, determined by genetic analysis of the MATP gene in a large Japanese population with OCA. Of 75 unrelated patients that were screened, 18 individuals (24%) were identified as having OCA4; they harbored seven novel mutations, including four missense mutations (P58S, D157N, G188V, and V507L) and three frameshift mutations (S90CGGCCA-->GC, V144insAAGT, and V469delG), showing that MATP is the most frequent locus for tyrosinase-positive OCA in Japanese patients. We discuss the functional melanogenic activity of each mutant allele, judging from the relationship between the phenotypes and genotypes of the patients. This is the first report on a large group of patients with OCA4.     

Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity

The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH.     

Mutations in the human orthologue of the mouse underwhite gene (uw) underlie a new form of oculocutaneous albinism, OCA4

Oculocutaneous albinism (OCA) affects approximately 1/20,000 people worldwide. All forms of OCA exhibit generalized hypopigmentation. Reduced pigmentation during eye development results in misrouting of the optic nerves, nystagmus, alternating strabismus, and reduced visual acuity. Loss of pigmentation in the skin leads to an increased risk for skin cancer. Two common forms and one infrequent form of OCA have been described. OCA1 (MIM 203100) is associated with mutations of the TYR gene encoding tyrosinase (the rate-limiting enzyme in the production of melanin pigment) and accounts for approximately 40% of OCA worldwide. OCA2 (MIM 203200), the most common form of OCA, is associated with mutations of the P gene and accounts for approximately 50% of OCA worldwide. OCA3 (MIM 203290), a rare form of OCA and also known as "rufous/red albinism," is associated with mutations in TYRP1 (encoding tyrosinase-related protein 1). Analysis of the TYR and P genes in patients with OCA suggests that other genes may be associated with OCA. We have identified the mouse underwhite gene (uw) and its human orthologue, which underlies a new form of human OCA, termed "OCA4." The encoded protein, MATP (for "membrane-associated transporter protein") is predicted to span the membrane 12 times and likely functions as a transporter.     

OCA4: evidence for a founder effect for the p.D157N mutation of the MATP gene in Japanese and Korean

Oculocutaneous albinism type 4 (OCA4) was identified as a rare form of human OCA among a group of autosomal recessive hypopigmentary disorders. Little is known about the prevailing distribution of patients of OCA4 with mutations of the MATP gene, although one Turkish, five German, one Korean, and 18 Japanese patients have been reported so far. The p.D157N mutation was previously reported to be the most frequent (0.389; 14/36) in Japanese patients and was also found in a Korean patient. On the other hand, this mutation has not been found in Turkish and German patients. We therefore investigated haplotypes of the patients who have the p.D157N mutation. The results showed that OCA4 is prevalent in East Asia including Japan and Korea likely as a result of a founder effect for the p.D157N mutation. Furthermore, it is suspected that the p.D157N mutation might have occurred on an ancestral chromosome after the divergence of Orientals and Caucasians.     

Oculocutaneous albinism with TYRP1 gene mutations in a Caucasian patient

Non-syndromic oculocutaneous albinism (OCA) is a clinically and genetically heterogeneous autosomal recessive disorder with mutations identified in several genes: OCA1 (tyrosinase, TYR), OCA2 (OCA2), OCA3 (tyrosinase-related protein 1, TYRP1), and OCA4 (membrane-associated transporter protein, MATP). OCA3 was thought to be restricted to black populations, where it was clinically described as rufous or brown albinism, until the recent report of a homozygous TYRP1 mutation in Caucasian patients from a consanguineous Pakistani family. Here, we describe a German patient of Caucasian origin, with a light-yellow skin, yellow-gold hair with orange highlights, fair eyelashes, several pigmented naevi, and no tendency to tan, only to burn. Eye-colour is blue-green with substance defects of the iris. Molecular analysis did not reveal any mutation in the TYR and OCA2 genes. Two mutations were found in the TYRP1 gene: a missense mutation (c.1066G>A/p.Arg356Glu) that was inherited from the mother, and a de novo single-base deletion (c.106delT/p.Leu36X). This finding suggests that mutation screening should be extended to the TYRP1 gene in patients from all ethnic origins, at least in cases where no mutations have been identified in the other OCA genes.     

Computational Screening of Disease-Associated Mutations in OCA2 Gene

Oculocutaneous albinism type 2 (OCA2), caused by mutations of OCA2 gene, is an autosomal recessive disorder characterized by reduced biosynthesis of melanin pigment in the skin, hair, and eyes. The OCA2 gene encodes instructions for making a protein called the P protein. This protein plays a crucial role in melanosome biogenesis, and controls the eumelanin content in melanocytes in part via the processing and trafficking of tyrosinase which is the rate-limiting enzyme in melanin synthesis. In this study we analyzed the pathogenic effect of 95 non-synonymous single nucleotide polymorphisms reported in OCA2 gene using computational methods. We found R305W mutation as most deleterious and disease associated using SIFT, PolyPhen, PANTHER, PhD-SNP, Pmut, and MutPred tools. To understand the atomic arrangement in 3D space, the native and mutant (R305W) structures were modeled. Molecular dynamics simulation was conducted to observe the structural significance of computationally prioritized disease-associated mutation (R305W). Root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessibility surface area, hydrogen bond (NH bond), trace of covariance matrix, eigenvector projection analysis, and density analysis results showed prominent loss of stability and rise in mutant flexibility values in 3D space. This study presents a well designed computational methodology to examine the albinism-associated SNPs.     

Oculocutaneous albinism type 4: six novel mutations in the membrane-associated transporter protein gene and their phenotypes

Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane-Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530-amino-acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene.     

Characterization of OCA2 cDNA in different porcine breeds and analysis of its potential effect on skin pigmentation in a red Iberian strain

Although the function of the OCA2 gene product has not been totally clarified, variation in OCA2 has been associated with skin and hair pigmentation in human and mouse. However, its contribution to skin colour in domestic species has not been reported. In this study, cDNA and intron 9 sequences of the porcine OCA2 gene have been characterized in several pig populations. The cDNA sequence alignment of 20 animals from eight porcine populations allowed the identification of 10 single nucleotide polymorphisms (SNPs); five of the 10 SNPs were non-synonymous. The intron 9 sequence alignment of 12 animals belonging to four pig populations revealed four additional SNPs. Skin colour variation was analysed in a red strain of Iberian pigs with segregation of three SNPs forming two OCA2 intragenic haplotypes. Results from this study provide evidence of a suggestive dominant effect of haplotypes on colour intensity and indicate an important contribution of additive polygenic effects (h2 = 0.56 +/- 0.21) to the variance of this trait.