Supplementary Material for: Identification of a splicing variant c.3813-3A>G in NPHP3 by reanalysis of whole exome sequencing in a Chinese boy with nephronophthisis

Nephronophthisis is an autosomal recessive cystic kidney disease characterized by tubular injury and commonly results in kidney failure. We reported a case of 4-year-old Chinese boy presented with severe anemia, kidney and liver dysfunction. Whole exome sequencing (WES) was performed to identify the candidate variant with a negative result initially. After complete collection of clinical information, reanalysis of WES identified a homozygous NPHP3 variant c.3813-3A>G (NM_153240.4). The effect on mRNA splicing of the intronic variant was predicted through software (three in silico splice tools). Furthermore, in vitro minigene assay was conducted to validate the predicted deleterious effects of the intronic variant. All of the splice prediction programs and minigene assay indicated that the variant had an impact on the normal splicing pattern of NPHP3. Our study confirmed the effect of the c.3813-3A>G variant on NPHP3 splicing in vitro, which gives additional evidence for the clinical significance of the variant and provides a basis for genetic diagnosis of nephronophthisis 3. In addition, we think that it is essential to reanalyze WES data after the complete clinical information collection to avoid missing some important candidate variants.

肾髓质囊性病（Nephronophthisis）是一类以肾小管损伤为特征的常染色体隐性遗传性囊性肾病，常可进展为肾衰竭。我们报告1例以重度贫血、肝肾功能异常为临床表现的4岁中国男性患儿。首先通过全外显子组测序（Whole exome sequencing, WES）筛选候选致病变异，初始分析结果为阴性。在完整收集患儿临床信息后，我们重新分析WES数据，检出1个纯合的NPHP3变异：c.3813-3A>G（NM_153240.4）。我们通过三款计算机辅助剪接预测工具（in silico splice tools），预测了该内含子变异对mRNA剪接的影响。此外，我们开展了体外迷你基因实验，以验证该内含子变异的潜在有害效应。所有剪接预测程序与迷你基因实验结果均显示，该变异可干扰NPHP3的正常剪接模式。本研究通过体外实验证实了c.3813-3A>G变异对NPHP3剪接的影响，为该变异的临床意义提供了额外证据，并为肾髓质囊性病3型的遗传学诊断提供了依据。此外，我们认为在完整收集临床信息后重新分析WES数据，对于避免遗漏重要候选致病变异至关重要。