Supplementary Material for: Biallelic TMEM72 variants in patients with a nephronophthisis-like phenotype

Introduction: Nephronophthisis (NPHP) is an autosomal recessive kidney disease resulting mainly from primary cilium defects, with unspecific and variable symptoms that can progress to kidney failure needing replacement therapy at a young age. Currently, up to 64% of likely NPHP cases can be diagnosed by assessing known genes. Therefore, there is a need to gain more insight in what genes can cause this disease.
Methods: In a diagnostic setting, we performed broad genetic testing in patients with advanced kidney disease. We carried out in silico and in vitro analyses for TMEM72, including immunohistochemistry and affinity proteomics, and in vivo experiments to further interpret our findings.
Results: We identified biallelic TMEM72 variants in nine patients from six families with a phenotype suggestive for NPHP. Five families presented with kidney failure at a (young) adult age. One family had a different phenotype with prenatal onset of kidney failure and neurological symptoms. The phenotypes of the patients correspond to TMEM72 expression mainly in the kidney. In silico analyses indicate that homozygous loss-of-function variants are likely not tolerated in TMEM72. Immunohistochemistry staining of kidney biopsies revealed altered localization and expression of TMEM72 in cases compared to controls. In human-derived tubuloids, we showed that TMEM72 localizes to the cilium. Furthermore, using an affinity proteomics approach, we found an association of TMEM72 and ciliary function, more specifically in selective ciliary cholesterol transport.
Conclusion: We present the first genetic evidence, underlined by immunohistochemistry and protein binding assays, linking TMEM72 variants to kidney disease and ciliary function. We conclude that TMEM72 is a candidate gene for NPHP. Future work is needed to further characterize TMEM72 variants and unravel its disease mechanism.

Introduction: 肾消耗病（Nephronophthisis, NPHP）是一种常染色体隐性遗传性肾病，主要由原发性纤毛缺陷引发，症状无特异性且表现多样，可在年轻时进展为肾衰竭，需接受替代治疗。目前，通过检测已知相关基因，仅能对至多64%的疑似NPHP病例作出诊断。因此，亟需深入解析可导致该病的致病基因。
Methods: 在临床诊断场景中，我们对患有进展性肾病的患者开展了广谱遗传学检测。针对TMEM72基因，我们开展了计算机模拟（in silico）与体外（in vitro）分析，涵盖免疫组化与亲和蛋白质组学研究，并通过体内（in vivo）实验进一步阐释本次研究结果。
Results: 我们在6个家系的9名表型疑似NPHP的患者中，检测到了TMEM72基因的双等位基因变异。其中5个家系的患者在（青年）成年阶段即出现肾衰竭。1个家系的患者表型存在差异，表现为产前起病的肾衰竭伴神经系统症状。患者的表型与TMEM72基因主要在肾脏中的表达模式相符。计算机模拟分析显示，TMEM72基因中纯合功能丧失型变异大概率无法被机体耐受。肾活检组织的免疫组化染色结果显示，与对照组相比，患者样本中TMEM72的定位与表达均发生异常改变。在人类源性肾小管类器官中，我们证实TMEM72定位于纤毛（cilium）。此外，通过亲和蛋白质组学方法，我们发现TMEM72与纤毛功能存在关联，具体涉及选择性纤毛胆固醇转运过程。
Conclusion: 本研究首次提供了遗传学证据，并通过免疫组化与蛋白质结合实验予以佐证，将TMEM72基因变异与肾病及纤毛功能异常建立了关联。我们认为TMEM72是NPHP的候选致病基因。后续仍需开展深入研究，以全面解析TMEM72基因变异的特征，并阐明其致病机制。