Hurtado Begoña, Muñoz Xavier, Mulero Maria Carme, Navarro Gemma, Domènech Pere, García de Frutos Pablo, Pérez-Riba Mercè, Sala Núria
Center de Genètica Mèdica i Molecular, IDIBELL, L'Hospitalet de Llobregat, Barcelona.
Haematologica. 2008 Apr;93(4):574-80. doi: 10.3324/haematol.12090. Epub 2008 Mar 5.
The molecular mechanisms by which PROS1 mutations result in protein S deficiency are still unknown for many of the mutations, particularly for those that result in a premature termination codon. The aim of this study was to analyze the functional relevance on mRNA and protein expression of 12 natural PROS1 mutations associated with protein S deficiency.
Five mutations were nonsense, three were small frameshift deletions, one was c.258,259AG>GT at the 3' end of exon 3, one was p.M640T and the last two were c.-7C>G and p.L15H, found in double heterozygosis as [c.-7C>G;44T>A]. The apparently neutral variant p.R233K was also analyzed. PROS1 cDNA was assessed by reverse transcriptase polymerase chain reaction of platelet mRNA. Expression of mutant proteins was determined by site-directed mutagenesis and analyses of transiently transfected PROS1 mutants in COS-7 cells.
Only cDNA from the normal allele was observed from the five nonsense mutations, the frameshift deletion c.1731delT and from c.258,259AG>GT. Both the normal and the mutated alleles were observed from [c.-7C>G;44T>A], c.187,188delTG and p.M640T. Transient expression analyses of PROS1 mutants whose mRNA was normally expressed revealed greatly reduced secretion of p.L15H and c.1272delA, mild secretion values of p.M640T and normal secretion levels of c.-7C>G and, as expected, p.R233K.
Whereas the main cause of quantitative protein S deficiency associated with missense mutations is defective synthesis, stability or secretion of the mutated protein, the main mechanism for the deficiency associated with mutations that generate a premature termination codon is not the synthesis of a truncated protein, but the exclusion of the mutated allele, probably by nonsense-mediated mRNA decay.
对于许多导致蛋白S缺乏的PROS1突变，尤其是那些导致提前终止密码子的突变，其导致蛋白S缺乏的分子机制仍不清楚。本研究的目的是分析与蛋白S缺乏相关的12种天然PROS1突变对mRNA和蛋白表达的功能相关性。
5种突变为无义突变，3种为小框移缺失，1种为外显子3 3'端的c.258,259AG>GT，1种为p.M640T，最后2种为c.-7C>G和p.L15H，以[c.-7C>G;44T>A]的双杂合形式存在。还分析了明显中性的变体p.R233K。通过血小板mRNA的逆转录聚合酶链反应评估PROS1 cDNA。通过定点诱变和对COS-7细胞中瞬时转染的PROS1突变体的分析来确定突变蛋白的表达。
从5种无义突变、框移缺失c.1731delT和c.258,259AG>GT中仅观察到来自正常等位基因的cDNA。从[c.-7C>G;44T>A]、c.187,188delTG和p.M640T中观察到正常和突变等位基因。对mRNA正常表达的PROS1突变体的瞬时表达分析显示，p.L15H和c.1272delA的分泌大大减少，p.M640T的分泌值中等，c.-7C>G和预期的p.R233K的分泌水平正常。
与错义突变相关的定量蛋白S缺乏的主要原因是突变蛋白的合成、稳定性或分泌缺陷，而与产生提前终止密码子的突变相关的缺乏的主要机制不是截短蛋白的合成，而是突变等位基因的排除，可能是通过无义介导的mRNA降解。
