Sequence alterations in the reduced folate carrier are observed in osteosarcoma tumor samples.
Sequence alterations in the reduced folate carrier are observed in osteosarcoma tumor samples.
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发表时间：
2003-02
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通讯作者：
Rui Yang;R. Sowers;B. Mazza;J. Healey;A. Huvos;H. Grier;M. Bernstein;G. Beardsley;M. Krailo;M. Devidas;J. Bertino;P. Meyers;R. Gorlick
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作者：
Rui Yang;R. Sowers;B. Mazza;J. Healey;A. Huvos;H. Grier;M. Bernstein;G. Beardsley;M. Krailo;M. Devidas;J. Bertino;P. Meyers;R. Gorlick
高剂量甲氨蝶呤是治疗高级别骨肉瘤的标准组成部分。其有效性可能受到内在和后天抗性的限制。在诊断时，大约一半的骨肉瘤中显示出还原型叶酸载体（RFC）表达减少。RFC基因的突变和多态性已在各种细胞系中报道。本研究的目的是调查骨肉瘤肿瘤样本中RFC基因的序列改变。通过DNA单链构象多态性筛选了162例骨肉瘤患者样本中RFC基因的整个编码区，然后对迁移率改变的任何区域进行直接测序。观察到先前鉴定的RFC外显子2的cDNA位置编号174的多态性。61例（37.6%）在该位置具有A/G杂合（His（27）/Arg（27）），52例（32.2%）具有G纯合（Arg（27）），49例（30.2%）具有A纯合（His（27））。15例（9.2%）样本在外显子2中鉴定出其他RFC序列变异，均未报告。外显子2中的序列变体包括cDNA位置231处的G至A置换、cDNA位置155处的G至A置换、cDNA位置114处的C至T置换和cDNA位置104处的T至C置换，导致氨基酸46处的丝氨酸至天冬酰胺置换、氨基酸21处的谷氨酸至赖氨酸置换、分别在氨基酸7处丙氨酸到缬氨酸的取代和在氨基酸4处丝氨酸到脯氨酸的取代。还观察到cDNA位置126处的A缺失导致移码。在多个样品中观察到其中一些变体。8个样本在外显子3中改变了单链构象多态性模式，这些模式与改变氨基酸序列的核苷酸变化相关。所有这些RFC序列变体似乎都是杂合的。在RFC cDNA外显子3的790位也观察到杂合C/T和纯合C，其不改变氨基酸编码序列。这项研究表明，RFC序列的改变是常见的骨肉瘤患者的样本。正在进行其他研究，以确定这些序列改变的临床意义及其对甲氨蝶呤转运和耐药性的影响。
High-dose methotrexate is a standard component of therapy for high-grade osteosarcoma. Its effectiveness may be limited by intrinsic and acquired resistance. Decreased reduced folate carrier (RFC) expression has been shown in approximately half of osteosarcomas at diagnosis. Mutations and polymorphisms in the RFC gene have been reported in various cell lines. The purpose of this study was to investigate sequence alterations in the RFC gene in osteosarcoma tumor samples. The entire coding region of the RFC gene in samples from 162 osteosarcoma patients was screened by DNA single-stranded conformational polymorphism, followed by direct sequencing of any region with altered mobility. A previously identified polymorphism at cDNA position number 174 of RFC exon 2 was observed. Sixty-one samples (37.6%) were heterozygous with both A/G at this position (His(27)/Arg(27)), 52 samples (32.2%) were homozygous with G (Arg(27)), and 49 samples (30.2%) were homozygous with A (His(27)). Fifteen (9.2%) samples were identified with other RFC sequence variants in exon 2, none of which have been reported. The sequence variants in exon 2 included a G to A substitution at cDNA position 231, a G to A substitution at cDNA position 155, a C to T substitution at cDNA position 114, and a T to C substitution at cDNA position 104, resulting in a serine to asparagine substitution at amino acid 46, a glutamate to lysine substitution at amino acid 21, an alanine to valine substitution at amino acid 7, and a serine to proline substitution at amino acid 4, respectively. A deletion of A at cDNA position 126 resulting in a frameshift was also observed. Some of these variants were observed in multiple samples. Eight samples had altered single-stranded conformational polymorphism patterns in exon 3 that were associated with nucleotide changes that altered the amino acid sequence. All of these RFC sequence variants appeared to be heterozygous. Heterozygous C/T and homozygous C also were observed at RFC cDNA position 790 in exon 3, which does not alter the amino acid coding sequence. This study shows that RFC sequence alterations are frequent in samples from osteosarcoma patients. Additional studies are under way to determine the clinical significance of these sequence alterations and their effect on methotrexate transport and resistance.