Background Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD-MUC1) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD-MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique.

Methods We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD-MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens.

Results The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD-MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients.

Conclusions Diagnosing ADTKD-MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD-MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.

背景技术为患有肾小管间质性肾病和继发性变性疾病（例如高血压）的患者提供正确的诊断仍然是一个挑战。由MUC1突变（ADTKD- MUC1）引起的常染色体显性肾小管间质性肾脏疾病（ADTKD）亚型特别难以诊断，因为突变热点是一个复杂的重复域，常规测序技术无法实现。在这里，我们进一步评估了SNaPshot微型测序作为诊断ADTKD- MUC1的技术，并评估了与疾病相关的粘蛋白1移码蛋白（MUC1-fs）的免疫检测作为非遗传技术。

方法我们通过在三个独立的ADTKD- MUC1家族中通过微卫星分析通过单倍型重建，通过靶向重复富集和SNaPshot微型测序重新评估了MUC1突变的检测。此外，我们生成了针对MUC1-fs的兔多克隆抗体，并评估了人肾活检标本中野生型和突变等位基因产物的免疫检测。

结果SNaPshot微型测序技术可检测MUC1突变。用我们的MUC1-fs抗体和MUC1抗体进行免疫染色显示，在人ADTKD- MUC1肾脏中都容易检测到这两种蛋白，粘蛋白1定位于心尖膜，而MUC1-fs大量分布在整个细胞质中。值得注意的是，临床肾脏样品中MUC1-fs表达的免疫组织化学分析有助于可靠地预测单个患者的疾病状态。

结论通过分子遗传学诊断ADTKD- MUC1是可能的，但技术要求高且劳动强度大。然而，对肾活检标本进行免疫组织化学对ADTKD- MUC1的非遗传诊断是可行的，因此，是选择家族进行进一步诊断的有效方法。我们的数据与MUC1-fs的特定分子作用是该病发病机理的假说相符。