原發性兒童腎病症候群之基因探討: 從臨床觀察到次世代基因定序研究

Abstract

背景 原發性兒童腎病症候群（Idiopathic Nephrotic Syndrome, INS）是一種常見的兒童腎臟疾病，其典型特徵包含蛋白尿(proteinuria)、低白蛋白血症 (hypoalbuminemia)、水腫(edema)與與高血脂 (hyperlipidemia)。目前對於INS的病因學仍然不完全清楚，但基因變異被認為是導致此病的關鍵因素之一。

研究目的 本研究旨在分析20例INS患兒的基因變異分析與判讀,通過次世代基因定序探索INS的基因變異特徵，以及這些變異對疾病發生、發展和治療反應的影響。

方法 研究對象為20例INS患兒，使用全外顯子定序分析對其基因進行了深入分析。並探討了AGT、APOL1、ANLN、CAPN12、COL4A3、COL4A4、CRB2、IL1RAP、INF2、LAMA5、MYO1E、NPHS1、NUP93、PODXL、SMARCAL1、SYNPO、TNS2 WDR73和等基因的變異與INS關連及臨床表徵之相關性。

結果 在進行20例INS患兒的基因變異分析中，本研究發現18位患兒帶有疾病相關的潛在變異，並確認四個顯著相關基因，其中單基因變異的檢出率達到20%。臨床反應觀察顯示，65%(13/20）的患兒對類固醇治療反應良好。本研究觀察到LAMA5是20例患兒中變異數目最多的基因（共四例），第二為CAPN12（共兩例）、NUP93（共兩例）、SYNPO（共兩例）。6位（30%）患兒帶有雙基因變異。攜帶LAMA5和CAPN12雙基因變異的患兒，表現出較早的發病年齡和較輕微的臨床症狀，且對藥物治療有更佳反應。然而，基於VarCoPP評估，這些雙重基因變異的致病性分數未顯示統計上顯著意義（99%信賴區間要求 VarCoPP 分數至少為0.647(hg38))，顯示其致病機轉尚需進一步研究。 在SRNS患者群體中，根據基因變異與臨床表現的關聯性，將變異分為三類(category1:確定有文獻報導過為致病點位，或已經很明確是疾病相關致病基因。category2:目前文獻未報導過，但不能排除和病人臨床症狀相關的罕見變異。category3:和臨床症狀相關的已知易感性變異或危險因子。），分別占總變異數的25%、62.5%及12.5%。相對的，在SSNS患者群體中，這三類變異則分別占總變異數的17.65%、76.47%及5.88%。此外，NUP93與SYNPO基因變異與SRNS的致病途徑相關。SYNPO基因變異的案例進一步凸顯了其在維持腎小球足細胞功能和結構的重要性，近期文獻顯示與糖尿病腎病變相關度高。 最後本研究也探討LAMA5與異位性皮膚炎（Atopic dermatitis, AD）的相關性時，發現帶有LAMA5變異的患者出現AD症狀，這可能顯示LAMA5在皮膚組織基底膜結構和功能中的關鍵作用。LAMA5變異可能影響皮膚屏障的完整性，加劇AD症狀，並潛在地與氣喘及其他過敏性疾病相關。此可能可以解釋INS病童常會合併有異位性體質的情況。

結論 本研究透過對20例原發性兒童腎病症候群（Idiopathic Nephrotic Syndrome, INS）患兒的基因變異分析，深入了解INS的基因變異特徵及其對疾病發生、發展和治療反應的影響。結果顯示，大部分患兒帶有至少一種與疾病相關的潛在變異，其中尤以LAMA5、CAPN12、NUP93和SYNPO等基因的變異較為常見。 此外，還發現INS患兒中存在不同類型的基因變異，這些變異根據其與疾病的相關性被分為三大類，分別是確定致病之單基因基因、雙基因以及已知易感性變異或危險因子。這一分類有助於更精確地理解INS的遺傳異質性，並為個體化治療提供參考。 值得注意的是，本研究發現攜帶LAMA5和CAPN12雙基因變異的患兒表現出較早的發病年齡和較輕微的臨床症狀，且對藥物治療有較好反應。然而，這些雙重基因變異的致病性分數在統計上並未達到顯著意義，因此其致病機轉仍需進一步研究。本研究還觀察到LAMA5基因變異與異位性皮膚炎（Atopic dermatitis, AD）的相關性，顯示LAMA5可能在皮膚組織的基底膜結構和功能中發揮重要作用。這一發現可理解INS病童常見的異位性體質，也為進一步探討腎病與皮膚疾病之間的相互作用有新的研究方向。

Background Idiopathic Nephrotic Syndrome (INS) is a prevalent kidney disease, characterized by proteinuria, hypoalbuminemia, generalized edema and hyperlipidemia. The etiology of INS is still not completely understood; however, genetic variations is considered to be a key factor in the onset of the disease.

Research Objective This study aims to analyze the genomes of 20 children with INS using next-generation sequencing to explore the genetic variation characteristics of INS and the impact of these variations on the onset, progression, and treatment response of the disease.

Methods The study involved 20 children with INS, for whom a deep analysis of their targeted genes was conducted using whole exome sequencing. The focus was on the variations of genes such as COL4A3, WDR73, MYO1E, APOL1, INF2, SMARCAL1, CRB2, PODXL, AGT, ADAMTS13, IL1RAP, NPHS1, ANLN, CAPN12, SYNPO, NUP93, COL4A4 and LAMA5.

Results In the genetic variation analysis of 20 children with INS, this study found that 18 of the children exhibited potential disease-related variations, and confirmed four significantly associated genes, with a detection rate of single-gene variations reaching 20%.Clinical response observations revealed that the vast majority patients responded well to steroid treatment. The study observed that LAMA5 had the highest number of variations among the 20 children (four cases), followed by CAPN12 (two cases), NUP93 (two cases), and SYNPO (two cases)。We also identified 6 children (30%) have digenic inheritance. Children carrying dual gene variations in LAMA5 and CAPN12 showed earlier onset of the disease and milder clinical symptoms, and had better response to drug treatment. However, based on VarCoPP assessment, the pathogenicity scores of these dual gene variations did not show statistical significance(To achieve a 99% confidence interval, the VarCoPP score must be at least 0.647(hg38。），indicating that further research is needed on their pathogenic mechanisms.

In the SRNS patient population, genetic variations are categorized into three groups based on their association with clinical manifestations. These groups include: category2: Variants not reported in the literature cannot be ruled out as rare variants related to the patient''s clinical symptoms. Category 3: Known susceptibility variants or risk factors associated with clinical symptoms. The factors account for 25%,62.5%, and 12.5% of the total variations, respectively. In contrast, within the SSNS patient population, these three types of variations account for 17.65%,76.47%,and 5.88% of the total variations, respectively. Additionally, variations in the NUP93 and SYNPO genes are associated with the pathogenic pathway of SRNS. The case of SYNPO gene variations further highlights its importance in maintaining the function and structure of glomerular podocytes. Recent literature shows a strong correlation with diabetic nephropathy. Finally, when exploring the relationship between LAMA5 and atopic dermatitis (AD), this study found that patients with LAMA5 variations exhibited symptoms of AD, possibly indicating a key role of LAMA5 in the structure and function of the skin basement membrane. LAMA5 variations may affect the integrity of the skin barrier, exacerbate AD symptoms, and potentially be associated with asthma and other allergic diseases. And this might explain most of the INS patients had atopy characteristics.

Conclusion This study conducted genetic variation analysis on 20 cases of idiopathic nephrotic syndrome (INS) in children to gain a deeper understanding of the genetic characteristics of INS and their impact on the occurrence, development, and treatment response of the disease. The results showed that the majority of the patients carried at least one potential variation associated with the disease, with variations in genes such as LAMA5, CAPN12, NUP93, and SYNPO being particularly significant. Furthermore, it was found that there are different types of genetic variations in INS patients, which were classified into three categories based on their relevance to the disease: definitive pathogenic monogenic condition, potentially digenic condition, and known susceptibility variations or risk factors. This classification helps to improve the understanding of the genetic heterogeneity of INS and serves as a reference for personalized treatment. It is worth noting that the study revealed that patients with dual gene variations in LAMA5 and CAPN12 exhibited an earlier onset age and milder clinical symptoms, as well as a better response to drug treatment. However, the pathogenicity scores of these dual gene variations did not reach statistical significance. Therefore, further research is needed to understand their pathogenic mechanisms. The study also examined the relationship between variations in the LAMA5 gene and atopic dermatitis (AD), suggesting that LAMA5 may have a significant impact on the structure and function of the basement membrane in skin tissue. This finding helps us understand the common atopic constitution in children with INS and opens up new research directions for further exploring the interaction between kidney disease and skin disorders.