Abstract：
BACKGROUND: Genomic studies have identified numerous genetic variants associated with susceptibility to multiple sclerosis (MS); however, each one explains only a small percentage of the risk of developing the disease. These variants are located in genes involved in specific pathways, which supports the hypothesis that the risk of developing MS may be linked to alterations in these pathways, rather than in specific genes. We analyzed the role of the TNFRSF1A gene, which encodes one of the TNF-α receptors involved in a signaling pathway previously linked to autoimmune disease.
METHODS: We included 138 individuals from 23 families including at least 2 members with MS, and analyzed the presence of exonic variants of TNFRSF1A through whole-exome sequencing. We also conducted a functional study to analyze the pathogenic mechanism of variant rs4149584 (-g.6442643C > G, NM_001065.4:c.362 G > A, R92Q) by plasmid transfection into human oligodendroglioma (HOG) cells, which behave like oligodendrocyte lineage cells; protein labeling was used to locate the protein within cells. We also analyzed the ability of transfected HOG cells to proliferate and differentiate into oligodendrocytes.
RESULTS: Variant rs4149584 was found in 2 patients with MS (3.85%), one patient with another autoimmune disease (7.6%), and in 5 unaffected individuals (7.46%). The 2 patients with MS and variant rs4149584 were homozygous carriers and belonged to the same family, whereas the remaining individuals presented the variant in heterozygosis. The study of HOG cells transfected with the mutation showed that the protein does not reach the cell membrane, but rather accumulates in the cytoplasm, particularly in the endoplasmic reticulum and near the nucleus; this suggests that, in the cells presenting the mutation, TNFRSF1 does not act as a transmembrane protein, which may alter its signaling pathway. The study of cell proliferation and differentiation found that transfected cells continue to be able to differentiate into oligodendrocytes and are probably still capable of producing myelin, although they present a lower rate of proliferation than wild-type cells.
CONCLUSIONS: Variant rs4149584 is associated with risk of developing MS. We analyzed its functional role in oligodendrocyte lineage cells and found an association with MS in homozygous carriers. However, the associated molecular alterations do not influence the differentiation into oligodendrocytes; we were therefore unable to confirm whether this variant alone is pathogenic in MS, at least in heterozygosis.
METHODS: We included 138 individuals from 23 families including at least 2 members with MS, and analyzed the presence of exonic variants of TNFRSF1A through whole-exome sequencing. We also conducted a functional study to analyze the pathogenic mechanism of variant rs4149584 (-g.6442643C > G, NM_001065.4:c.362 G > A, R92Q) by plasmid transfection into human oligodendroglioma (HOG) cells, which behave like oligodendrocyte lineage cells; protein labeling was used to locate the protein within cells. We also analyzed the ability of transfected HOG cells to proliferate and differentiate into oligodendrocytes.
RESULTS: Variant rs4149584 was found in 2 patients with MS (3.85%), one patient with another autoimmune disease (7.6%), and in 5 unaffected individuals (7.46%). The 2 patients with MS and variant rs4149584 were homozygous carriers and belonged to the same family, whereas the remaining individuals presented the variant in heterozygosis. The study of HOG cells transfected with the mutation showed that the protein does not reach the cell membrane, but rather accumulates in the cytoplasm, particularly in the endoplasmic reticulum and near the nucleus; this suggests that, in the cells presenting the mutation, TNFRSF1 does not act as a transmembrane protein, which may alter its signaling pathway. The study of cell proliferation and differentiation found that transfected cells continue to be able to differentiate into oligodendrocytes and are probably still capable of producing myelin, although they present a lower rate of proliferation than wild-type cells.
CONCLUSIONS: Variant rs4149584 is associated with risk of developing MS. We analyzed its functional role in oligodendrocyte lineage cells and found an association with MS in homozygous carriers. However, the associated molecular alterations do not influence the differentiation into oligodendrocytes; we were therefore unable to confirm whether this variant alone is pathogenic in MS, at least in heterozygosis.
摘要：
背景：基因组研究已经确定了许多与多发性硬化症（MS）易感性相关的遗传变异；然而，每一个都只解释了一小部分患这种疾病的风险。这些变异位于参与特定途径的基因中，这支持了发展MS的风险可能与这些途径的改变有关的假设，而不是特定的基因。我们分析了TNFRSF1A基因的作用，它编码参与先前与自身免疫性疾病相关的信号通路的TNF-α受体之一。
方法：我们纳入了来自23个家庭的138名个体，其中包括至少2名患有MS的成员，并通过全外显子组测序分析了TNFRSF1A外显子变体的存在。我们还进行了一项功能研究，以分析变体rs4149584（-g.6442643C>G，NM_001065.4:c.362G>A，R92Q）通过质粒转染人少突胶质细胞瘤（HOG）细胞，其表现类似少突胶质细胞谱系细胞;蛋白质标记用于定位细胞内的蛋白质。我们还分析了转染的HOG细胞增殖和分化成少突胶质细胞的能力。
结果：在2例MS患者中发现了rs4149584变体（3.85％），一名患有另一种自身免疫性疾病的患者（7.6％），5个未受影响的个体（7.46%）。2例MS患者和变异体rs4149584为纯合携带者，属于同一家族，而其余的个体在杂合中呈现变异。对转染突变的HOG细胞的研究表明，该蛋白未到达细胞膜，而是在细胞质中积累，特别是在内质网和核附近；这表明，在呈现突变的细胞中，TNFRSF1不作为跨膜蛋白，这可能会改变其信号通路。对细胞增殖和分化的研究发现，转染的细胞继续能够分化为少突胶质细胞，并且可能仍然能够产生髓鞘，尽管它们的增殖率低于野生型细胞。
结论：变体rs4149584与发展为MS的风险相关。我们分析了其在少突胶质细胞谱系细胞中的功能作用，并在纯合携带者中发现了与MS的关联。然而,相关的分子改变不影响分化为少突胶质细胞;因此，我们无法证实这种变异是否单独在MS中致病，至少在杂合方面。
方法：我们纳入了来自23个家庭的138名个体，其中包括至少2名患有MS的成员，并通过全外显子组测序分析了TNFRSF1A外显子变体的存在。我们还进行了一项功能研究，以分析变体rs4149584（-g.6442643C>G，NM_001065.4:c.362G>A，R92Q）通过质粒转染人少突胶质细胞瘤（HOG）细胞，其表现类似少突胶质细胞谱系细胞;蛋白质标记用于定位细胞内的蛋白质。我们还分析了转染的HOG细胞增殖和分化成少突胶质细胞的能力。
结果：在2例MS患者中发现了rs4149584变体（3.85％），一名患有另一种自身免疫性疾病的患者（7.6％），5个未受影响的个体（7.46%）。2例MS患者和变异体rs4149584为纯合携带者，属于同一家族，而其余的个体在杂合中呈现变异。对转染突变的HOG细胞的研究表明，该蛋白未到达细胞膜，而是在细胞质中积累，特别是在内质网和核附近；这表明，在呈现突变的细胞中，TNFRSF1不作为跨膜蛋白，这可能会改变其信号通路。对细胞增殖和分化的研究发现，转染的细胞继续能够分化为少突胶质细胞，并且可能仍然能够产生髓鞘，尽管它们的增殖率低于野生型细胞。
结论：变体rs4149584与发展为MS的风险相关。我们分析了其在少突胶质细胞谱系细胞中的功能作用，并在纯合携带者中发现了与MS的关联。然而,相关的分子改变不影响分化为少突胶质细胞;因此，我们无法证实这种变异是否单独在MS中致病，至少在杂合方面。
