Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

中文翻译：

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使用定量熔解分析检测脆性 X 综合征和 X 染色体非整倍体中的偏斜 X 染色体失活

脆性 X 智力低下的甲基化 1 (FMR1) 外显子 1/内含子 1 边界位于脆性 X 相关表观遗传元件 2 (FREE2)，揭示了脆性 X 综合征全突变 (FM: CGG > 200) 女性中的偏斜 X 染色体失活 (XCI)。XCI 偏斜也与异常的 X 连锁基因表达有关，对性染色体非整倍体 (SCA) 具有更广泛的临床影响。在这项研究中，使用甲基化特异性定量熔解分析 (MS-QMA) 靶向了 10 个 FREE2 CpG 位点，其中包括 3 个无法用以前使用的 EpiTYPER 系统分析的位点。该方法用于检测 FM 女性和不同类型 SCA 中的偏斜 XCI。我们测试了从 107 名对照（CGG < 40）、148 名 FM 和 90 名 SCA 个体收集的静脉血和唾液 DNA。MS-QMA 确定： (i) 如果结合 Y 染色体测试，大多数 SCA；(ii) 基因座特异性 XCI 在 FM 雌性中偏向低甲基化状态；(iii) 在具有 3 个或更多 X 染色体的 SCA 中，以及在 47,XXY 个体中的 5% 中，XCI 偏向于高甲基化状态。MS-QMA 输出与 FM 男性和女性的 EpiTYPER 参考方法也显示出显着相关性（磷< 0.0001) 和 SCA (磷< 0.05)。总之，我们展示了使用 MS-QMA 在两个具有诊断实用程序的应用程序中量化偏斜 XCI。