角化性皮肤病是一类以皮肤角化过程异常为特征的疾病，其病理机制主要涵盖表皮细胞异常增生引发角化加速、角质形成细胞终末分化紊乱导致角化过度，以及角质脱落障碍致使角质层相对增厚等多方面因素。多数情况下，其发病根源与遗传因素紧密相关。本研究聚焦于长岛型掌跖角化病，开展了初步的体外治疗实验探究，并针对一例全新的综合征型鱼鳞病明确了致病基因，旨在进一步深入剖析遗传性皮肤病的发病机制，并为精准治疗策略提供实验依据与理论支撑。

一、抑制性tRNA对长岛型掌跖角化病的体外治疗初探

背景：长岛型掌跖角化病（Nagashima-type palmoplantar keratosis, NPPK）是一种常染色体隐性遗传的非综合征性弥漫性掌跖角化病，由Nagashima首次提出并描述，其特征是边界清楚的弥漫性红斑性角化过度，延伸到掌跖背侧表面及跟腱区域，并常伴有掌跖部多汗症。在中国NPPK患者群体庞大，预计达数十万人，其中，75.9%患者携带SERPINB7基因c.796C>T，p.Arg266*(UGA)无义突变。对于此类提前终止密码子（premature termination codons, PTCs）所致疾病，目前尚无有效的临床治疗方案。

目的：本研究旨在明确抑制性 tRNA（suppressor-tRNA, sup-tRNA）在体外环境中，是否对 NPPK 最常见的致病基因 SERPINB7 位点 c.796C>T，p.Arg266*(UGA) 具有通读治疗作用，为治疗该疾病提供新的理论依据和潜在策略。

方法：

1.通过对天然 tRNA^Arg反密码子进行精准的点突变改造，成功构建出能够特异性通读UGA的sup-tRNA^Arg；

2.在mCherry质粒中构建UAG和UAA两种非靶标无义突变，检测sup-tRNA^Arg是否通读UGA之外的其他终止密码子。

3.在角质形成细胞系HaCaT中共转染SERPINB7 c.796C>T质粒和sup-tRNA^Arg质粒，通过蛋白免疫印记实验和酶活性测定实验，深入探究sup-tRNA^Arg是否能通读c.796C>T位点；

4.构建稳定表达SERPINB7 WT和SERPINB7 c.796C>T两种基因型的293T和HeLa细胞系，进一步探究sup-tRNA^Arg在不同人类细胞系背景下，针对稳定表达 SERPINB7 的通读效能。

5.分别通过Cell-Titer Glo试剂盒和实时荧光定量 PCR（qPCR）技术，检测sup-tRNA^Arg对HaCaT细胞活力的影响，以及其对M1极化状态下的THP-1细胞免疫应答的诱导作用，以此初步评估 sup-tRNA 疗法在体外环境中的安全性。

结果：

1.sup-tRNA^Arg显示出高度特异性，仅能通读终止密码子 UGA，对于终止密码子 UAA 或 UAG 无通读现象；

2.在转染 SERPINB7 c.796C>T 质粒的 HaCaT 细胞系中，sup-tRNA^Arg能够促使完整的 SERPINB7 蛋白重新表达，并且有效恢复了 SERPINB7 的酶活性；

3.在稳定表达SERPINB7 c.796C>T的293T和HeLa细胞系中，sup-tRNA^Arg同样展现出通读能力，可以恢复SERPINB7正常蛋白的表达；

4.本研究所用体外治疗剂量下，sup-tRNA^Arg未对 HaCaT 细胞活力产生显著的降低作用，亦不能显著诱导M1极化后THP-1的免疫应答。

结论：本研究证实，在体外环境中，sup-tRNA^Arg 能够有效通读SERPINB7 c.796C>T这一PTC位点，并恢复异常SERPINB7的表达和功能。sup-tRNA^Arg的通读作用具有特异性，仅针对UGA终止密码子有通读作用，且在实验条件下未观察到显著细胞毒性或免疫应答诱导作用。本研究为后续sup-tRNA^Arg对NPPK在体层面的通读治疗研究奠定了坚实的理论和实验基础，为 NPPK 的治疗开辟了新的潜在途径，有望推动该疾病治疗策略的发展与突破。

二、一种新的综合征型鱼鳞病的致病基因分析

背景：常染色体隐性先天性鱼鳞病（autosomal recessive congenital ichthyosis , ARCI）是一组具有遗传异质性的角化障碍，其典型特征为皮肤呈现鱼鳞状鳞屑。当鱼鳞病合并其他系统症状时，则被归类为综合征型鱼鳞病（Syndromic ichthyoses, SI）。鱼鳞病的致病基因复杂多样，精准识别患者的致病位点对于明确诊断、深入探究疾病机制以及开发针对性治疗手段具有关键意义。

目的：本研究旨在描述国内首例伴羊毛状发的先天性鱼鳞病病例，并深入探究其基因变异情况，以期丰富对综合征型鱼鳞病的认识并拓展其致病基因谱。

方法：收集先证者临床资料，采集外周血，提取基因组DNA，利用全外显子组测序技术进行检测，筛查可疑的致病基因，并经生物信息学分析后明确致病变异。进一步对致病位点进行PCR扩增和Sanger测序以验证变异情况。

结果：经全外显子组测序及一系列分析流程，发现先证者的PRSS8基因第3号外显子存在纯合变异c.124dupC，该变异将引起PRSS8蛋白的氨基酸出现移码变异（p. Gln42Profs*24）。根据 ACMG指南，结合患者临床表现，可初步判断该变异为致病性变异。

结论：PRSS8基因的纯合变异c.124dupC（p.Gln42Profs*24）是导致此例患者出现先天性鱼鳞病伴有羊毛状发的根本原因。该病例为国内首例、国际第2例由PRSS8基因变异导致的全新综合征型鱼鳞病，我们初步建议将其命名为“鱼鳞病-羊毛状发综合征”，同时强调了蛋白酶对于表皮屏障和毛囊发育的重要作用。

Keratinization disorders, a category of skin diseases marked by abnormal keratinization, primarily involve the abnormal hyperplasia of epidermal cells triggering accelerated keratinization, the disordered terminal differentiation of keratinocytes leading to hyperkeratosis, and the impaired corneocyte shedding causing relative hyperkeratosis. Genetics often underlie their etiology. This study centers on Nagashima palmoplantar keratosis (NPPK), probing its in-vitro treatment, and identifying the pathogenic gene for a novel syndromic ichthyoses (SI) case, to deepen the understanding of genetic skin disease mechanisms and underpin precise treatment strategies.

1. Preliminary In Vitro Investigation of Suppressor tRNA for Treating Nagashima-Type Palmoplantar Keratosis

Background: Nagashima-type palmoplantar keratosis (NPPK) is an autosomal recessive non-syndromic diffuse palmoplantar keratoderma, first described by Nagashima. It is characterized by well-demarcated diffuse erythematous hyperkeratosis extending to the dorsal surfaces of the palms/soles and the Achilles tendon area, often accompanied by palmoplantar hyperhidrosis. In China, the NPPK patient population is substantial, estimated at hundreds of thousands, with 75.9% harboring the SERPINB7 c.796C>T, p.Arg266 (UGA)* nonsense mutation. Currently, there are no effective clinical treatments for diseases caused by premature termination codons (PTCs).

Objective: This study aims to determine whether suppressor tRNA (sup-tRNA) can mediate readthrough of the most common NPPK-causing mutation, SERPINB7 c.796C>T (UGA), in vitro, providing new theoretical insights and potential therapeutic strategies.

Methods:

1. Engineered a UGA-specific sup-tRNA^Arg via precise anticodon modification of native tRNA^Arg.

2. Constructed non-target nonsense mutations (UAG, UAA) in an mCherry plasmid to assess sup-tRNA^Arg specificity.

3. Co-transfected SERPINB7 c.796C>T plasmid and sup-tRNA^Arg into HaCaT keratinocytes, evaluating readthrough efficacy via Western blot and enzymatic activity assays.

4. Established stable 293T and HeLa cell lines expressing either SERPINB7 WT or c.796C>T to validate sup-tRNA^Arg readthrough capability across cell types.

5. Assessed safety by measuring HaCaT viability (Cell-Titer Glo) and THP-1 (M1-polarized) immune responses (qPCR).

Results:

1. sup-tRNA^Arg exhibited high specificity, readthrough only UGA, not UAA or UAG.

2. Restored full-length SERPINB7 protein expression and enzymatic activity in HaCaT cells.

3. Demonstrated consistent readthrough in 293T and HeLa cells stably expressing SERPINB7 c.796C>T.

4. No significant cytotoxicity or immune activation observed at experimental doses.

Conclusion: sup-tRNA^Arg effectively mediates UGA-specific readthrough of SERPINB7 c.796C>T, rescuing protein expression and function without detectable toxicity. This study lays a foundation for future in vivo investigations and offers a novel therapeutic avenue for NPPK.

2. Pathogenic Gene Analysis of a Novel Syndromic Ichthyosis

Background: Autosomal recessive congenital ichthyosis (ARCI) encompasses genetically heterogeneous keratinization disorders characterized by fish-like scaling. When accompanied by systemic manifestations, it is classified as syndromic ichthyosis (SI). Given the genetic complexity of ichthyoses, precise identification of pathogenic variants is critical for diagnosis, mechanistic studies, and targeted therapies.

Objective: To characterize the first Chinese case of congenital ichthyosis with woolly hair and identify its genetic basis, expanding the understanding of SI-associated genes.

Methods: Collected clinical data and peripheral blood from the proband. Genomic DNA was subjected to whole-exome sequencing (WES), followed by bioinformatic filtering and validation via Sanger sequencing.

Results: WES revealed a homozygous PRSS8 c.124dupC variant in exon 3, causing a frameshift (p.Gln42Profs*24). Based on ACMG guidelines and clinical presentation, this variant was classified as pathogenic.

Conclusion: The PRSS8 c.124dupC variant underlies this novel "ichthyosis-woolly hair syndrome", representing the first Chinese and second global case linked to PRSS8. Our findings highlight the role of proteases in epidermal barrier and hair follicle development.