Ohmiya N, Matsumoto S, Yamamoto H, Baranovskaya S, Malkhosyan S R, Perucho M
The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Gene. 2001 Jul 11;272(1-2):301-13. doi: 10.1016/s0378-1119(01)00517-0.
Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.
具有微卫星突变体表型（MMP）的遗传性和散发性胃肠道癌的特征是在简单重复序列处存在显著的基因组不稳定。这种基因组不稳定通常由DNA错配修复（MMR）基因hMSH2和hMLH1中的种系和体细胞突变引起。MMP也可能由hMLH1的表观遗传失活导致。MMP在含有单核苷酸重复序列的基因中产生许多体细胞移码突变。我们之前报道过，在MMP肿瘤中，hMSH6和hMSH3 MMR基因通常分别在其（C）（8）和（A）（8）序列中携带移码突变。我们提出这些“继发性突变体突变”有助于MMP的逐渐显现。在此，我们报告在MMP的结肠癌和胃癌中检测到这些基因中的其他移码、无义及错义突变。在一个携带另一个体细胞移码突变的结肠肿瘤中，发现hMSH6存在种系移码突变。在hMSH6中检测到几个种系序列变异和保守残基处的体细胞错义突变，而在hMSH3中仅检测到一个。在hMSH6保守残基处的三个种系变异中，一个与（C）（8）序列处的体细胞突变共存，另一个有体细胞错义突变。我们认为其中一些种系和体细胞错义变异具有致病性。虽然在一些肿瘤中发现了双等位基因hMSH6和hMSH3移码突变，但许多肿瘤似乎仅含有单等位基因突变。在一些肿瘤中，发现这些（C）（8）和（A）（8）序列处的体细胞单等位基因移码突变与另一个等位基因中的其他体细胞突变共存，这支持了它们在肿瘤发生过程中的功能。然而，hMSH6和hMSH3中这些额外体细胞突变的低发生率使得许多肿瘤仅含有单等位基因突变。通过对不同基因型的不同肿瘤细胞克隆中的RNA和蛋白质表达进行比较分析，研究了移码突变对基因表达的影响。结果表明，hMSH6（C）（8）移码突变消除了蛋白质表达，排除了截短蛋白的显性负效应。我们在累积单倍体不足模型的背景下提出这些继发性单等位基因突变体突变的功能。
