背景：长岛型掌跖角化症（NPPK）是一种常染色体隐性遗传性皮肤病，是东亚人群中最常见的遗传性掌跖角化病的类型。临床表现为掌跖部位境界清楚的红斑伴弥漫性角化过度。2013年，Kubo等人首次报告了SERPINB7的双等位基因功能丧失突变是NPPK的根本原因。2022年，我们建立了中国最大的NPPK队列并发现该病在我国的患病率约为0.975/10,000。其中大部分NPPK患者明确了SERPINB7双等位基因的突变。然而，部分呈现NPPK典型表现的患者，基因检测结果却仅提示SERPINB7的单等位基因变异。此外，NPPK目前仍无有效治疗手段。
目的：本研究旨在探究仅携带SERPINB7单等位基因变异的NPPK患者中的深内含子变异及其致病性，揭示其分子机制，并尝试反义寡核苷酸（ASO）治疗，为NPPK患者开辟新治疗路径。
方法：
1. 探究NPPK队列中，携带SERPINB7单等位基因变异的NPPK患者中的深内含子变异情况：1）收集携带SERPINB7单等位基因变异的NPPK患者的临床资料及基因检测信息。2）通过定制化二代测序检测SERPINB7的深内含子变异。3）利用Sanger测序对患者变异位点进行共分离验证。
2. 探究NPPK患者中SERPINB7基因深内含子变异的致病性：1）苏木素-伊红染色和免疫荧光染色检测皮损组织中病理变化情况。2）提取患者掌部皮肤RNA，通过逆转录、PCR扩增、TA克隆和Sanger测序评估SERPINB7转录本的剪接改变。3）构建过表达异常剪接转录本的质粒，利用Western blot分析异常蛋白表达，并通过酶活性实验探究突变对蛋白功能的影响。4）进行单倍型分析以明确深内含子突变的始祖效应。
3. 探究SERPINB7基因深内含子变异引起异常剪接的机制：1）使用SpliceAid数据库预测异常剪接转录本中的RNA靶点基序和结合的剪接因子蛋白。2）RNA pull-down实验结合质谱分析检测差异表达的剪接因子蛋白。3）通过Western blot实验验证质谱分析中发现的差异表达剪接因子蛋白。
4. 探究ASO如何纠正SERPINB7深内含子变异引起的异常剪接：1）构建minigene，体外分析SERPINB7深内含子突变引起的异常剪接。2）设计靶向SERPINB7深内含子变异的ASO。3）体外共转染突变型minigene和ASO，以纠正异常剪接。
结果：
1. 在31例携带SERPINB7单等位基因变异的NPPK患者中，检测到7例患者同时携带SERPINB7 c.745-553T>G深内含子变异。
2. 这些患者的临床表现和病理改变与先前关于NPPK患者的研究结果一致。
3. 患者掌部皮肤RNA分析结果显示，在SERPINB7的pre-mRNA序列中，第7外显子和第8外显子之间因该变异的发生而插入了一个含90个核苷酸的7号内含子片段，最终导致了假外显子包含的异常剪接现象。
4. Western blot和酶活性实验表明，该变异产生了截短且失功能的SERPINB7蛋白，其抑制Legumain酶活性的能力降低。
5. 单倍型分析表明SERPINB7 c.745-553T>G为中国人群中的始祖突变。
6. 剪接因子蛋白SRSF9通过与该变异创建的内含子剪接增强子元件（ISE）“UGGAU”结合，激活了隐匿的剪接位点，促进了假外显子的包含。
7. ASO治疗在体外有效逆转了SERPINB7 c.745-553T>G导致的假外显子包含。
结论：本研究首次证实SERPINB7 c.745-553T>G在NPPK中的致病性。该变异引发了假外显子包含的异常剪接，产生了截短且失功能的SERPINB7蛋白。同时，该变异被证明是中国人群中的始祖突变。研究还发现SRSF9剪接因子蛋白与变异产生的ISE结合，激活隐匿剪接位点，促进了假外显子包含。此外，本研究通过体外转染ASO成功纠正了异常剪接，恢复了SERPINB7的正常转录，为NPPK治疗提供了理论依据。

Background: Nagashima-type palmoplantar keratoderma (NPPK), an autosomal recessive inherited skin disease, is the most common type of hereditary palmoplantar keratosis in the East Asian population. The clinical manifestation is well-defined erythema on the palms and soles, accompanied by mild diffuse hyperkeratosis. The histopathology shows mild to moderate hyperkeratosis accompanied by mild perivascular inflammatory infiltration. Kubo et al. first identified biallelic loss-of-function variants in SERPINB7 as the underlying cause of NPPK in 2013. In 2022, we established the largest NPPK research cohort and found that the prevalence was approximately 0.975/10,000 in China. The majority of them were found to have identified biallelic SERPINB7 variants. However, some patients exhibiting typical NPPK features had only monoallelic variant in SERPINB7. At present, there is still no effective treatment for NPPK.
Objective: This study aims to identify a deep intronic splicing variant and its pathogenicity in NPPK patients carrying only monoallelic variant in SERPINB7, explore its molecular mechanisms, and evaluate a targeted antisense oligonucleotide (ASO) therapy.
Methods: 
1. Exploration of the deep intronic variant in patients with NPPK carrying only monoallelic variant in SERPINB7 in the NPPK cohort: 1) Collect the clinical data and genetic testing information of NPPK patients carrying only monoallelic variant in SERPINB7. 2) Detect deep intronic variants in the SERPINB7 gene through customized next-generation sequencing 3) Use Sanger sequencing to verify the co-segregation of variants in patients.
2. Investigation of the pathogenicity of deep intronic variants in the SERPINB7 gene in NPPK patients: 1) Detect pathological changes in lesional tissues by hematoxylin-eosin staining and immunofluorescence staining. 2) Extract RNA from the palm skin of patients, and evaluate the splicing changes of SERPINB7 through reverse transcription, PCR amplification, TA cloning, and Sanger sequencing. 3) Construct plasmids overexpressing abnormally spliced transcripts, analyze abnormal protein expression by Western blot, and explore the effect of the variant on protein function through enzyme activity assays. 4) Perform haplotype analysis to clarify the founder effect of the deep intronic variant.
3. Exploration of the mechanism of abnormal splicing caused by the deep intronic variant in the SERPINB7 gene: 1) Use the SpliceAid database to predict RNA target motifs and splicing factor proteins in abnormally spliced transcripts. 2) Detect expressed splicing factor proteins by RNA pull-down assay combined with mass spectrometry analysis. 3) Validate the differentially expressed splicing factor proteins found in mass spectrometry analysis by Western blot.
4. Research on how antisense oligonucleotides (ASO) correct the abnormal splicing caused by the deep intronic variant in the SERPINB7 gene: 1) Construct minigenes to analyze the aberrant splicing caused by the deep intronic variant in SERPINB7 in vitro. 2) Design ASOs targeting the deep intronic variant in the SERPINB7 gene. 3) Co-transfect the mutant minigene and ASO in vitro to correct the abnormal splicing.
Results: 
1. Among 31 NPPK patients carrying only monoallelic variant in SERPINB7, 7 patients were detected with the SERPINB7 c.745-553T>G deep intronic variant.
2. The clinical manifestations and pathological changes of these patients were consistent with the previous research results on NPPK.
3. Analysis of RNA from the palm skin of patients demonstrated that this variant led to the insertion of a 90-bp from intron 7 between exon 7 and exon 8, resulting in the pseudo-exon inclusion.
4. Western blot and enzyme activity assay showed that the variant produced a truncated and dysfunctional SERPINB7 protein with reduced ability to inhibit Legumain enzyme activity.
5. Haplotype analysis indicated that SERPINB7 c.745-553T>G was a founder variant in the Chinese population.
6. The splicing factor protein SRSF9 bound to the intronic splicing enhancer element (ISE) “UGGAG” created by this variant, activating the cryptic splice site and promoting pseudo-exon inclusion.
7. ASO therapy effectively corrected the pseudo-exon inclusion caused by SERPINB7 c.745-553T>G in vitro.
Conclusion: This study established the pathogenicity of a deep intronic variant in SERPINB7, which causes aberrant splicing and results in a truncated and dysfunctional SERPINB7 protein. Meanwhile, this variant was proven to be a founder variant in the Chinese population. SRSF9 was identified as a key regulator of this abnormal splicing. It can bind to the ISE generated by the variant, activating the cryptic splice site and promoting the inclusion of the pseudo-exon. Furthermore, this study successfully corrected the aberrant splicing through in vitro ASO therapy, providing a theoretical foundation for the treatment of NPPK.