Genetic Analysis Of Klotho In Diseases Of Aging

根据对klotho突变小鼠的研究，klotho基因在抑制几种不同的衰老表型方面发挥了作用。Klotho基因缺陷纯合的小鼠表现出一种与人类衰老非常相似的症状，包括动脉粥样硬化、骨质疏松症、肺气肿、不孕不育和皮肤萎缩。我们已经在杰克逊实验室的一组特征良好的小鼠品系中寻找了klotho基因的序列变异。Klotho基因的每个外显子都在这20个菌株中进行了测序。这项工作现在提交给了《基因组学》杂志。具体地说，我们发现：1.在该小组的16个实验室来源的近交系中没有发现任何变异。2.在4个野生自交系中发现了45个变异，包括43个单核苷酸替换、1个缺失和1个插入。在核苷酸替换中，有6个导致了氨基酸替换。3.实时荧光定量RT-PCR分析结果表明，SPRET/EI菌株的Klotho基因表达水平高于其他野生菌株和实验室菌株。4.3株氨基酸取代野生型菌株的Klotho mRNA膜/分泌型比值均高于对照菌株。 我们发现野生衍生物种SPRET/EI的klotho mRNA的表达水平大约是实验室衍生菌株的两倍，并且有四个氨基酸变化，这是有趣的，因为SPRET/EI和实验室衍生菌株之间的几个表型差异可能与klotho表达有关。这些表型差异包括寿命长、听力异常、总胆固醇低和高密度脂蛋白水平。有趣的是，Molf/EI在野生来源的菌株中表现出最低的klotho表达水平，在小鼠基因组计划中测试的43种菌株中，其总胆固醇水平最高，而高密度脂蛋白百分比最低。 我们推测，Klotho mRNA的改变和SPRET/EI表达水平的增加可能对与年龄相关的疾病提供保护，如听力损失和冠状动脉疾病。还有一种可能是，小鼠身上特定的klotho变异可能与延长寿命有关，就像在人类身上看到的那样。 我们的IRB批准的在巴尔的摩老龄化纵向研究参与者中寻找Klotho基因功能变异的方案扩大到也包括Inchianti人群。我们已经对整个BLSA和Inchianti人群的KL-VS等位基因进行了基因分型。数据分析正在进行中，以寻找BLSA和INCHIANTI研究中检查的特定等位基因变异和表型之间的相关性。在过去的一年里，我们扩大了分析范围，包括BLSA和INCHIANTI人群中LMNA基因的多态。 我们刚刚完成了巴尔的摩AING纵向研究中Klotho与多态之间的关系的分析。我们发现，在男性中，这种多态与胰岛素抵抗(异常的2小时血糖托伦斯试验)有关，而在女性中则没有。有趣的是，在男性和女性中，VS+基因多态与空腹胰岛素无关。这与之前的研究一致，这些研究无法在不同代表性人群中发现Klotho基因多态与空腹血糖或通过空腹血糖异常诊断出的糖尿病之间的关系。Inchianti研究中关于Klotho多态的数据尚未分析。

The klotho gene is known to play a role in suppressing several different aging phenotypes, based on studies in the klotho mutant mouse. Mice homozygous for defects in the klotho gene exhibit a syndrome that closely resembles human aging, including atherosclerosis, osteoporosis, emphysema, infertility and skin atrophy. We have looked for sequence variation in the klotho gene in a paenl of well-characterized mouse strains from the Jackson Laboratory. Each exon of the klotho gene was sequenced in each of these 20 strains.This work is now being submitted to the journal Genomics. Specifically, we have found: 1. No variation was found in any of the 16 laboratory derived inbred stains in the panel. 2.Among the 4 wild-derived inbred strains, 45 variants were found, including 43 single nucleotide substitutions, one deletion and one insertion. Of the nucleotide substitutions, six resulted in amino acid substitutions. 3.Real-time RT-PCR analysis of klotho gene expression in the wild-derived strains has shown a higher level of gene expression in SPRET/Ei than in the other wild-derived or laboratory-derived strains. 4. The ratio of membrane form to secreted form of klotho mRNA is higher in the three wild-derived strains with amino acid substitutions that in the control strains. Our finding that the klotho mRNA in the wild-derived species SPRET/Ei is expressed at approximately twice the level of laboratory derived strains and has four amino acid changes is intriguing in light of several phenotypic differences between SPRET/Ei and laboratory derived strains that may be related to klotho expression. These phenotypic differences include a long life-span, exceptional hearing, low total cholesterol and high HDL levels. Interestingly, MOLF/Ei, which exhibited the lowest klotho expression levels of the wild-derived strains, had the highest total cholesterol levels and lowest percent HDL of the 43 strains tested in the mouse phenome project. We hypothesize that klotho mRNA alterations and increased expression levels in SPRET/Ei may provide protection against age-related diseases such as hearing loss and coronary artery disease. It is also possible that specific klotho variants in mice may be associated with increased longevity, as has been seen in humans. Our IRB-approved protocol to look for functional variants of the Klotho gene among participants in the Baltimore Longitudinal Study on Aging was expanded to include the InChianti population as well. We have genotyped the entire BLSA and InChianti populations for the KL-VS allele. Data analysis is underway to search for correlations between specific allelic variants and phenotypes examined in the BLSA and InChianti studies. Over the past year, we have expanded the analysis to include polymorphisms in the LMNA gene in both the BLSA and InCHIANTI populations. We have just completed the analysis of the relationship between the Klotho VS polymorphism in the Baltimore Longitudinal Study of Aing. We found that the polymorpism is associated with insulin resistance (abnormal 2h glucose tollerance test) in men but not in women. Interestingly, both in men and in women, the VS+ polymorphism was not associated with fasting insulin. This is consistent with previous studies that were unable to finds a relationship between Klotho polymorphism and fasting glucose or diabetes diagnosed through abnormal fasting glucose in different representative populations. The data on the Klotho polymorphism in the InCHIANTI study have not been analyzed.