BACKGROUND The MUC1 gene is associated with autosomal dominant tubulointerstitial kidney disease (ADTKD), leading to CKD. Current methods of sequencing, like exome sequencing, rarely detect MUC1 pathogenic variants because of the variable number of tandem repeats (VNTR) in MUC1 exon2. We demonstrated that combining fast read filtering with a sensitive VNTR genotyping strategy enables systematic screening of 27dupC pathogenic MUC1 variant from exome data. METHODS We initially validated our bioinformatics pipeline in a proof-of-concept cohort incorporating exome data from 33 participants with a known MUC1 pathogenic variant identified by Snapshot PCR and confirmed by 54 MUC1-negative individuals for negative control. We then retrospectively analyzed exome sequencing data from January 2019 to October 2023 from 3512 adult participants with nephropathy of unknown origin. Finally, we prospectively validated our pipeline in 825 additional participants enrolled from November 2023. RESULTS SharkVNTyper accurately identified MUC1 variants in 32 out of 33 participants and excluded its presence in all the 54 negative controls in the proof-of-concept cohort (sensitivity of 97%, specificity of 100%). Integration of Shark tool with VNTyper significantly reduced running time from 6-12 hours to 5-10 minutes per sample, allowing both retrospective and prospective analysis. In the retrospective cohort, SharkVNTyper identified 23 additional positive cases that were not suspected clinically and had been missed in the initial exome analysis; 18 of these cases were confirmed as carrying the MUC1 27dupC mutation by low-throughput Snapshot PCR. In the prospective cohort of 825 CKD cases, systematic screening discovered 13 positive cases, with 12 confirmed by PCR. Overall, of 63 participants (1.4% of 4653) with molecularly confirmed ADTKD-MUC1, comprehensive diagnoses and descriptions of the disease were available for 24 participants. Median age of kidney failure was 50 years, 38% exhibited bilateral multiple kidney cysts, 8% had early-onset gout, and 58% had arterial hypertension. CONCLUSIONS SharkVNTyper enables the analysis of highly repeated regions, such as the MUC1 VNTR, and facilitates the systematic screening of ADTKD-MUC1 from exome data, fostering 27dupC variation identification.

中文翻译：

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通过外显子组测序系统筛选常染色体显性肾小管间质肾病 - MUC1 27dupC 致病性变异。


背景 MUC1 基因与常染色体显性遗传性肾小管间质肾病 （ADTKD） 相关，导致 CKD。目前的测序方法，如外显子组测序，由于 MUC1 外显子 2 中的串联重复序列 （VNTR） 数量可变，因此很少检测到 MUC1 致病性变异。我们证明，将快速读取过滤与敏感的 VNTR 基因分型策略相结合，可以从外显子组数据中系统筛选 27dupC 致病性 MUC1 变体。方法 我们最初在一个概念验证队列中验证了我们的生物信息学管道，该队列结合了来自 33 名参与者的外显子组数据，这些参与者具有通过 Snapshot PCR 鉴定的已知 MUC1 致病性变异，并由 54 名 MUC1 阴性个体确认为阴性对照。然后，我们回顾性分析了 2019 年 1 月至 2023 年 10 月的 3512 名不明原因肾病成年参与者的外显子组测序数据。最后，我们在 2023 年 11 月开始招募的 825 名额外参与者中前瞻性地验证了我们的管道。结果 SharkVNTyper 在 33 名参与者中的 32 名中准确识别了 MUC1 变异，并在概念验证队列的所有 54 名阴性对照中排除了它的存在（敏感性为 97%，特异性为 100%）。Shark 工具与 VNTyper 的集成将每个样品的运行时间从 6-12 小时显著缩短到 5-10 分钟，从而允许进行回顾性和前瞻性分析。在回顾性队列中，SharkVNTyper 确定了另外 23 例临床上未怀疑且在初始外显子组分析中遗漏的阳性病例;其中 18 例通过低通量快照 PCR 证实携带 MUC1 27dupC 突变。在 825 例 CKD 病例的前瞻性队列中，系统筛查发现 13 例阳性病例，其中 12 例经 PCR 确诊。总体而言，在 63 名参与者 （1.4% 的 4653） 分子证实的 ADTKD-MUC1,24 名参与者可以获得疾病的全面诊断和描述。肾衰竭的中位年龄为 50 岁，38% 表现为双侧多发性肾囊肿，8% 为早发性痛风，58% 为动脉高血压。结论 SharkVNTyper 能够分析高度重复的区域，例如 MUC1 VNTR，并有助于从外显子组数据中系统筛选 ADTKD-MUC1，促进 27dupC 变异鉴定。