Laboratório de Medicina Genômica and Centro de Genética Médica-Serviço de Referência em Doenças Raras/IFF/FIOCRUZ, Rio de Janeiro, Brasil.
Unidade de Genética Clínica, Centro de Genética Médica-Serviço de Referência em Doenças Raras/IFF/FIOCRUZ, Rio de Janeiro, Brasil.
Genet Test Mol Biomarkers. 2021 Oct;25(10):674-682. doi: 10.1089/gtmb.2020.0330.
Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are related to skeletal dysplasias (SDs): acondroplasia (ACH), hypochodroplasia (HCH) and type I (TDI) and II (TDII) tanatophoric dysplasias. This study was designed to standardize and implement a high-resolution melting (HRM) technique to identify mutations in patients with these phenotypes. Initially, gene segments from 84 patients were PCR amplified and subjected to Sanger sequencing. Samples from 29 patients positive for mutations were analyzed by HRM. Twelve of the patients mutations had ACH (six g.16081 G > A, three g.16081 G > C and three g.16081 G > A + g.16002 C > T); thirteen of patients with HCH had mutations (eight g.17333 C > A, five g.17333 C > G and five were negative); and four patients with DTI had mutations (three g.13526 C > T and one g.16051G > T and two patients with DTII (presented mutation g.17852 A > G). When analyzing the four SDs altogether, an overlap of the dissociation curves was observed, making genotyping difficult. When analyzed separately, however, the HRM analysis method proved to be efficient for discriminating among the mutations for each SD type, except for those patients carrying additional polymorphism concomitant to the recurrent mutation. We conclude that for recurrent mutations in the gene, that the HRM technique can be used as a faster, reliable and less expensive genotyping routine for the diagnosis of these pathologies than Sanger sequencing.
成纤维细胞生长因子受体 3（FGFR3）基因突变与骨骼发育不良（SDs）有关：软骨发育不全（ACH）、软骨发育不全（HCH）、I 型（TDI）和 II 型（TDII）致死性发育不良。本研究旨在标准化并实施高分辨率熔解（HRM）技术，以鉴定具有这些表型的患者中的突变。最初，对 84 例患者的基因片段进行 PCR 扩增，并进行 Sanger 测序。对 29 例突变阳性患者的样本进行 HRM 分析。12 例 ACH 患者的突变（6 例 g.16081 G > A、3 例 g.16081 G > C 和 3 例 g.16081 G > A + g.16002 C > T）；13 例 HCH 患者的突变（8 例 g.17333 C > A、5 例 g.17333 C > G 和 5 例阴性）；4 例 DTI 患者的突变（3 例 g.13526 C > T 和 1 例 g.16051G > T，2 例 DTII 患者（携带突变 g.17852 A > G）。当分析这四种 SD 时，观察到解离曲线的重叠，使基因分型变得困难。然而，当分别分析时，HRM 分析方法被证明对每种 SD 类型的突变具有区分能力，除了那些同时伴有复发性突变的附加多态性的患者。我们得出结论，对于 FGFR3 基因中的反复突变，HRM 技术可作为比 Sanger 测序更快、更可靠且更经济的基因分型常规方法，用于诊断这些疾病。
