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Abstract：
BACKGROUND: Pathogenic (P)/likely pathogenic (LP) SMAD3 variants cause Loeys-Dietz syndrome type 3 (LDS3), which is characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with osteoarthritis.
OBJECTIVE: Investigate the impact of P/LP SMAD3 variants with functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs), to optimize interpretation of SMAD3 variants.
METHODS: A retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and functional analyses on SMAD3 patient-derived VSMCs and SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts.
RESULTS: Individuals with dominant negative (DN) SMAD3 variant in the MH2 domain exhibited more major events (66.7% vs. 44.0%, P = 0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. The age at first major event was 35.0 years [IQR 29.0-47.0] in individuals with DN variants in MH2, compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (P = 0.065). Fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential in HI SMAD3 variant fibroblasts. HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix formation compared to control cell lines.
CONCLUSIONS: Distinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and SMA and MYH11 expression. The differences between DN and HI SMAD3 variant fibroblasts and VSMCs potentially contribute to the differences in disease manifestation. Notably, myofibroblast differentiation seems a suitable alternative in vitro test system compared to VSMCs.
OBJECTIVE: Investigate the impact of P/LP SMAD3 variants with functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs), to optimize interpretation of SMAD3 variants.
METHODS: A retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and functional analyses on SMAD3 patient-derived VSMCs and SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts.
RESULTS: Individuals with dominant negative (DN) SMAD3 variant in the MH2 domain exhibited more major events (66.7% vs. 44.0%, P = 0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. The age at first major event was 35.0 years [IQR 29.0-47.0] in individuals with DN variants in MH2, compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (P = 0.065). Fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential in HI SMAD3 variant fibroblasts. HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix formation compared to control cell lines.
CONCLUSIONS: Distinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and SMA and MYH11 expression. The differences between DN and HI SMAD3 variant fibroblasts and VSMCs potentially contribute to the differences in disease manifestation. Notably, myofibroblast differentiation seems a suitable alternative in vitro test system compared to VSMCs.
摘要：
背景：致病性（P）/可能致病性（LP）SMAD3变异导致Loeys-Dietz综合征3型（LDS3），以动脉瘤为特征，整个血管系统的解剖和弯曲与骨关节炎合并。
目的：研究P/LPSMAD3变体与功能测试对患者来源的成纤维细胞和血管平滑肌细胞（VSMC）的影响，优化SMAD3变体的解释。
方法：对具有P/LPSMAD3变体的个体的临床数据进行回顾性分析，并对SMAD3患者来源的VSMC和SMAD3患者来源的成纤维细胞进行功能分析，分化为肌成纤维细胞。
结果：MH2域中具有显性阴性（DN）SMAD3变体的个体表现出更多的主要事件（66.7％vs.44.0%,P=0.054),与单倍体不足（HI）变体相比，发生在更年轻的年龄。在MH2中具有DN变体的个体中，第一次重大事件的年龄为35.0岁[IQR29.0-47.0]，而在具有HI变体的个体中，年龄为46.0岁[IQR40.0-54.0]（P=0.065）。携带DNSMAD3变体的成纤维细胞显示出降低的分化潜能，与HISMAD3变体成纤维细胞的分化潜力增加相反。HISMAD3变体VSMC显示SMA表达升高和替代MYH11亚型的表达改变。与对照细胞系相比，DNSMAD3变体肌成纤维细胞显示出减少的细胞外基质形成。
结论：可以通过评估分化潜能来区分P/LPHI和DNSMAD3变异体，SMA和MYH11表达。DN和HISMAD3变体成纤维细胞和VSMC之间的差异可能导致疾病表现的差异。值得注意的是,与VSMC相比，肌成纤维细胞分化似乎是一种合适的体外测试系统。
目的：研究P/LPSMAD3变体与功能测试对患者来源的成纤维细胞和血管平滑肌细胞（VSMC）的影响，优化SMAD3变体的解释。
方法：对具有P/LPSMAD3变体的个体的临床数据进行回顾性分析，并对SMAD3患者来源的VSMC和SMAD3患者来源的成纤维细胞进行功能分析，分化为肌成纤维细胞。
结果：MH2域中具有显性阴性（DN）SMAD3变体的个体表现出更多的主要事件（66.7％vs.44.0%,P=0.054),与单倍体不足（HI）变体相比，发生在更年轻的年龄。在MH2中具有DN变体的个体中，第一次重大事件的年龄为35.0岁[IQR29.0-47.0]，而在具有HI变体的个体中，年龄为46.0岁[IQR40.0-54.0]（P=0.065）。携带DNSMAD3变体的成纤维细胞显示出降低的分化潜能，与HISMAD3变体成纤维细胞的分化潜力增加相反。HISMAD3变体VSMC显示SMA表达升高和替代MYH11亚型的表达改变。与对照细胞系相比，DNSMAD3变体肌成纤维细胞显示出减少的细胞外基质形成。
结论：可以通过评估分化潜能来区分P/LPHI和DNSMAD3变异体，SMA和MYH11表达。DN和HISMAD3变体成纤维细胞和VSMC之间的差异可能导致疾病表现的差异。值得注意的是,与VSMC相比，肌成纤维细胞分化似乎是一种合适的体外测试系统。
