Abstract：
Various genetic variants have been found to be associated with the clinical onset of premature ovarian insufficiency (POI). However, when measured in vitro, the functional influence of the variants can be difficult to determine. By whole-exome sequencing (WES) of 93 patients with sporadic POI, we found a missense variant c.623G > A;p.R208H in the EIF4ENIF1 gene. In silico prediction of the variant using different algorithms suggested it might be a damaging variant. We compared the property of EIF4ENIF1 R208H and Q842P, a POI-related mutant that we reported previously, with wildtype (WT) protein using 293FT cells in vitro. Surprisingly, a change in subcellular distribution and granule forming ability (Q842P) and nuclear import capacity (R208H) was not observed, despite domain prediction evidences. Since EIF4ENIF1 was reported to inhibit translation, we employed T&T-seq, a translation-transcription dual-omics sequencing method, to profile gene expression upon overexpression of EIF4ENIF1 WT and mutants. EIF4ENIF1 WT overexpression group exhibited significantly (P < 0.0001) lower translation efficiency (TE) than empty vector or GFP overexpression control group. Surprisingly, EIF4ENIF1 Q842P overexpression failed to repress global translation, showing an overall TE significantly higher than WT group. Overexpression R208H significantly (P < 0.0001) lowered the overall TE, whereas exhibiting a reduced translation inhibitory effect on high-TE genes (TE > 2 in GFP control group). Several fertility-associated genes, such as AMH in Q842P group and SERPINE1 and THBS1 in R208H group, was translationally up-regulated in mutant groups versus WT control, suggesting a potential mechanism of mutated EIF4ENIF1 causing POI via impaired translation repression. It is further proposed that T&T-seq can be a sensitive evaluation tool for the measurement of functional alteration by variants in many other translational regulator genes, not only EIF4ENIF1, helping to eliminate misinterpretation of clinical significance of genetic variants.
摘要：
已发现各种遗传变异与过早卵巢功能不全（POI）的临床发作有关。然而,当在体外测量时，变体的功能影响可能难以确定。通过对93例散发性POI患者的全外显子组测序（WES），我们发现了一个错义变体c.623G>A；p。R208H中的EIF4ENIF1基因。使用不同算法对变体进行计算机模拟预测表明，它可能是破坏性变体。我们比较了EIF4ENIF1R208H和Q842P的性质，我们之前报道的POI相关突变体，在体外使用293FT细胞与野生型(WT)蛋白。令人惊讶的是,未观察到亚细胞分布和颗粒形成能力（Q842P）和核输入能力（R208H）的变化，尽管有领域预测证据。由于据报道EIF4ENIF1抑制翻译，我们雇佣了T&Tseq,翻译-转录双组测序方法，在EIF4ENIF1WT和突变体过表达时描述基因表达。EIF4ENIF1WT过表达组表现出比空载体或GFP过表达对照组显著(P<0.0001)更低的翻译效率(TE)。令人惊讶的是,EIF4ENIF1Q842P过表达未能抑制全球翻译，显示总TE显著高于WT组。过表达R208H显著(P<0.0001)降低总TE,而对高TE基因表现出降低的翻译抑制作用(GFP对照组中TE>2)。几个与生育相关的基因，如Q842P组的AMH和R208H组的SERPINE1和THBS1，与WT对照相比，在突变体组中翻译上调，提示EIF4ENIF1突变通过翻译抑制受损引起POI的潜在机制。进一步提出，T＆T-seq可以是一种灵敏的评估工具，用于测量许多其他翻译调节基因中变体的功能改变。不仅EIF4ENIF1，有助于消除对遗传变异的临床意义的误解。
