CLOVES syndrome (OMIM# 612918) is a rare overgrowth disorder resulted from mosaic gain-of-function mutations in the PIK3CA gene. All the reported CLOVES-associated PIK3CA mutations are missense mutations affecting certain residues. We aim to investigate underlying mutation and its pathogenicity in a patient with CLOVES syndrome and to evaluate the inhibitory effects of the PI3K/AKT/mTOR pathway inhibitors. We performed whole-exome sequencing (WES) and Sanger sequencing to detect underlying somatic mutations in the skin lesion of the patient. Quantitative real-time PCR (qRT-PCR) was employed to evaluate the mRNA abundance of PIK3CA in the patient’s skin lesion. AKT phosphorylation level assessed by immunoblotting of lysates from transiently transfected cells was performed to evaluate the PIK3CA mutations and inhibitory effects of PI3K/AKT/mTOR pathway inhibitors. A somatic frameshift mutation c.3206_3207insG (p.X1069Trpfs*4) in PIK3CA was identified in the genomic DNA extracted from the vascular malformation sample of the patient. This mutation affects the canonical stop codon of PIK3CA (NM_006218.4) and is predicted to produce a prolonged protein with four additional residues. qRT-PCR demonstrated that the mRNA expression levels of the patient’s affected skin tissue were comparable compared to the normal control. In vitro studies revealed that p.X1069Trpfs*4 mutant exhibited increased AKT phosphorylation significantly to that of the wildtype, which could be inhibited by PI3K/AKT/mTOR pathway inhibitors. We have identified the first frameshift mutation in PIK3CA that causes CLOVES syndrome, which was confirmed to overactive PI3K/AKT/mTOR pathway by transient transfection assays. We also provided more evidence of ARQ092 to be a potential therapeutic option for PROS in vitro.

中文翻译：

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PIK3CA 体细胞移码突变通过激发 PI3K/AKT/mTOR 通路导致 CLOVES 综合征

CLOVES 综合征 (OMIM# 612918) 是一种罕见的过度生长疾病，由 PIK3CA 基因中的镶嵌功能获得性突变引起。所有报道的丁香相关 PIK3CA 突变都是影响某些残基的错义突变。我们旨在研究 CLOVES 综合征患者的潜在突变及其致病性，并评估 PI3K/AKT/mTOR 通路抑制剂的抑制作用。我们进行了全外显子组测序 (WES) 和 Sanger 测序，以检测患者皮肤病变中的潜在体细胞突变。采用定量实时 PCR (qRT-PCR) 来评估患者皮肤病变中 PIK3CA 的 mRNA 丰度。通过对瞬时转染细胞的裂解物进行免疫印迹评估 AKT 磷酸化水平，以评估 PIK3CA 突变和 PI3K/AKT/mTOR 通路抑制剂的抑制作用。在从患者血管畸形样本中提取的基因组 DNA 中鉴定出 PIK3CA 中的体细胞移码突变 c.3206_3207insG (p.X1069Trpfs*4)。该突变影响 PIK3CA (NM_006218.4) 的规范终止密码子，预计会产生具有四个额外残基的延长蛋白质。qRT-PCR 表明，与正常对照相比，患者受影响皮肤组织的 mRNA 表达水平相当。体外研究表明，p.X1069Trpfs*4 突变体表现出比野生型显着增加的 AKT 磷酸化，这可能被 PI3K/AKT/mTOR 通路抑制剂抑制。我们已经确定了导致 CLOVES 综合征的 PIK3CA 中的第一个移码突变，通过瞬时转染分析证实其过度活跃 PI3K/AKT/mTOR 通路。我们还提供了更多证据表明 ARQ092 是体外 PROS 的潜在治疗选择。