COMPUTATIONAL & LABORATORY STUDY OF P16/INK4 MUTATIONS

描述(申请人提供)：错义碱基替换可能或不可能 会导致蛋白质的功能改变。解读生物效应 在癌症相关基因中氨基酸(AA)的替换在 各种背景。临床癌症遗传学家经常必须决定一种 以前未知的等位基因变异会导致疾病。可靠的功能分析 通常是不可用的。拟议工作的目标是开发 并测试一个预测生物后果和临床后果的模型 通过对p16基因错义突变的详细研究 进化的替代模式和蛋白质结构。统一的AA和 核苷酸序列经常被比较以推断有关蛋白质的信息 功能。很少对基因进行详细的计算分析。 与人类疾病有关，但初步数据表明它们可以改善 在预测功能上的简单序列比对。具体目标是：1) 收集足以完善模型的p16进化和突变数据 它预测了AA取代的功能后果；克隆和 对新的p16序列进行测序，以便扩大数据库，使其变得更大 足以进行足够详细的计算。2)体外细胞检测 P16错义变异体的周期阻断和cdk结合。Logistic回归 将被用来建立预测功能丧失的计算模型。 高守恒性正确预测突变函数的初步计算 在75%-80%的测试密码子中，低保守性正确预测了 野生型的功能在85%-90%。我们预测进化参数 结构特征为预测提供了独立的信息 功能变化。3)将计算和实验室数据整合到 P16及相关蛋白的生物学鉴别。P16被用作 因其在家族性黑色素瘤中的作用而用于研究的原型；误解 发生功能后果未知的突变；可靠的功能 化验是存在的；而且晶体结构是已知的，所以突变的数据 光谱、进化、结构和功能可以相互关联。这些研究 应该可以推广到解释其他基因的突变 发现癌症相关基因和其他单核苷酸多态(SNPs) 在整个基因组中。

DESCRIPTION (provided by applicant): Missense base substitutions may or may not result in a functionally altered protein. Interpreting the biological effects of amino acid (AA) substitutions in cancer-related genes is critical in a variety of contexts. Clinical cancer geneticists often must decide whether a previously unknown allelic variant causes disease. Reliable functional assays usually are not available. The objectives of the proposed work are to develop and test a model for predicting the biological consequences and clinical relevance of missense mutations in the p16 gene by detailed study of evolutionary substitution patterns and protein structure. Aligned AA and nucleotide sequences are often compared to infer information about protein function. Detailed computational analyses are rarely performed on genes associated with human diseases, but preliminary data indicate they can improve upon simple sequence alignment in predicting function. Specific aims are: 1) To collect p16 evolutionary and mutational data sufficient for refining a model that predicts the functional consequences of AA substitutions; to clone and sequence new p16 sequences in order to expand the database so that it is large enough for sufficiently detailed computations. 2) To test in vitro the cell cycle arrest and cdk binding of missense variants of p16. Logistic regression will be used to establish a computational model that predicts loss of function. Initial calculations of high conservation correctly predicted mutant function in 75-80 percent of tested codons, and low conservation correctly predicted wild type function in 85-90 percent. We predict that evolutionary parameters and structural features provide independent information for predicting functional changes. 3) To integrate computational and laboratory data in differentiating the biology of p16 and related proteins. p16 is used as a prototype for study because of its role in Familial Melanoma; missense mutations occur whose functional consequences are unknown; reliable functional assays exist; and a crystal structure is known, so data for mutational spectrum, evolution, structure, and function can be correlated. These studies should be generalizable to the interpretation of mutations in other cancer-related genes and to other single nucleotide polymorphisms (SNPs) found throughout the genome.