Molecular Diagnosis of Mosaic Overgrowth Syndromes Using a Custom-Designed Next-Generation Sequencing Panel
Molecular Diagnosis of Mosaic Overgrowth Syndromes Using a Custom-Designed Next-Generation Sequencing Panel
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DOI：
10.1016/j.jmoldx.2017.04.006
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发表时间：
2017-07-01
影响因子：
4.1
通讯作者：
Li, Marilyn M.
中科院分区：
文献类型：
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作者：
Chang, Fengqi;Liu, Liu;Li, Marilyn M.
近年来的研究发现，先天性脂肪瘤过度生长伴血管、表皮和骨骼异常（CLOVES）综合征、Proteus综合征、巨脑-毛细血管畸形-多小回畸形（MCAP）综合征等一类过度生长综合征是由参与磷脂酰肌醇3-激酶/AKT/雷帕霉素通路机制靶基因的体细胞激活变异引起的。由于这些变异的低丰度性质，Sanger测序通常产生阴性结果。我们已经开发并验证了针对与这些综合征相关的所有已知变异的下一代测序（NGS）面板。50例，包括两例产前病例，使用该小组进行了测试。在50例中有28例发现了PIK3CA、PIK3R2或AKT1基因的致病变异，变异等位基因频率从1.0%到49.2%不等。在大多数情况下，这些变异只存在于受影响的组织中，表明在这些疾病的发展中起着因果作用。体外细胞培养显示，携带变异等位基因的细胞显著富集，表明这些变异使突变细胞具有生长优势。表型-基因型相关分析显示，PIK3CA残基E542、E545和H1047的突变热点常与CLOVES综合征相关，而PIK3CA G914R则优先与MCAP相关。因此，我们证明了NGS技术对检测低水平嵌合具有高度敏感性，可以促进产前和产后嵌合过度生长综合征的临床诊断。
Recent studies have discovered a group of overgrowth syndromes, such as congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) syndrome, Proteus syndrome, and megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, are caused by somatic activating variants in genes involved in the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway. Because of the low-abundance nature of these variants, Sanger sequencing often yields negative results. We have developed and validated a next-generation sequencing (NGS) panel that targets all known variants associated with these syndromes. Fifty cases, including two prenatal cases, were tested using the panel. A pathogenic variant in the PIK3CA, PIK3R2, or AKT1 gene was identified in 28 of the 50 cases with the variant allele frequencies ranging from 1.0% to 49.2%. These variants were only present in the affected tissues in most of the cases, demonstrating a causal role in the development of these diseases. In vitro cell culture showed significant enrichment of the cells harboring variant alleles, suggesting that these variants render growth advantages to mutant cells. Phenotype-genotype correlation analysis showed PIK3CA mutation hotspots at residues E542, E545, and H1047 are often associated with CLOVES syndrome, whereas PIK3CA G914R is preferentially related to MCAP. We thus demonstrate that NGS technology is highly sensitive for detecting Low-level mosaicism and can facilitate clinical diagnosis of mosaic overgrowth syndromes in both prenatal and postnatal settings.