Popp Nicholas A, Powell Rachel L, Wheelock Melinda K, Holmes Kristen J, Zapp Brendan D, Sheldon Kathryn M, Fletcher Shelley N, Wu Xiaoping, Fayer Shawn, Rubin Alan F, Lannert Kerry W, Chang Alexis T, Sheehan John P, Johnsen Jill M, Fowler Douglas M
Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA.
Medical Scientist Training Program, University of Washington School of Medicine, Seattle, WA, USA.
bioRxiv. 2025 Jan 29:2024.04.01.587474. doi: 10.1101/2024.04.01.587474.
Despite widespread advances in DNA sequencing, the functional consequences of most genetic variants remain poorly understood. Multiplexed Assays of Variant Effect (MAVEs) can measure the function of variants at scale, and are beginning to address this problem. However, MAVEs cannot readily be applied to the ~10% of human genes encoding secreted proteins. We developed a flexible, scalable human cell surface display method, Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP), to measure secreted protein variant effects. We used MultiSTEP to study the consequences of missense variation in coagulation factor IX (FIX), a serine protease where genetic variation can cause hemophilia B. We combined MultiSTEP with a panel of antibodies to detect FIX secretion and post-translational modification, measuring a total of 44,816 effects for 436 synonymous variants and 8,528 of the 8,759 possible missense variants. 49.6% of possible missense variants impacted secretion, post-translational modification, or both. We also identified functional constraints on secretion within the signal peptide and for nearly all variants that caused gain or loss of cysteine. Secretion scores correlated strongly with FIX levels in hemophilia B and revealed that loss of secretion variants are particularly likely to cause severe disease. Integration of the secretion and post-translational modification scores enabled reclassification of 63.1% of variants of uncertain significance in the hemophilia genotyping project. Lastly, we showed that MultiSTEP can be applied to a wide variety of secreted proteins. Thus, MultiSTEP is a multiplexed, multimodal, and generalizable method for systematically assessing variant effects in secreted proteins at scale.
尽管DNA测序技术取得了广泛进展，但大多数基因变异的功能后果仍知之甚少。变异效应多重检测（MAVEs）可以大规模测量变异的功能，并开始解决这一问题。然而，MAVEs不能轻易应用于约10%编码分泌蛋白的人类基因。我们开发了一种灵活、可扩展的人类细胞表面展示方法——细胞外蛋白多重表面拴系法（MultiSTEP），以测量分泌蛋白变异效应。我们使用MultiSTEP研究凝血因子IX（FIX）错义变异的后果，FIX是一种丝氨酸蛋白酶，其基因变异可导致B型血友病。我们将MultiSTEP与一组抗体相结合，以检测FIX的分泌和翻译后修饰，共测量了436个同义变异和8759个可能的错义变异中的8528个变异的44816种效应。49.6%的可能错义变异影响分泌、翻译后修饰或两者皆有。我们还确定了信号肽内以及几乎所有导致半胱氨酸增减的变异在分泌方面的功能限制。分泌评分与B型血友病患者的FIX水平密切相关，表明分泌变异的缺失尤其可能导致严重疾病。分泌和翻译后修饰评分的整合使得血友病基因分型项目中63.1%的意义未明变异能够重新分类。最后，我们表明MultiSTEP可应用于多种分泌蛋白。因此，MultiSTEP是一种用于大规模系统评估分泌蛋白变异效应的多重、多模态且可推广的方法。
