Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

尽管基因组技术和分析取得了重大进展，但 >50% 的神经发育障碍 （NDD） 患者在经过广泛评估后仍未得到诊断。例如，我们的临床异质性 NDD 患者队列在 FRAXA 检测、染色体微阵列分析和三重外显子组测序 （ES） 后仍未确诊。在这项研究中，我们探讨了男性患者和受影响女性的母亲中非随机 X 染色体失活 （XCI） 的频率，其基本原理是偏斜的 XCI 可能掩盖了以前在 X 染色体上发现的先前丢弃的遗传变异。采用基于多重荧光 PCR 的测定法分析用 HhaI 甲基化敏感的限制性内切酶消化后的 XCI 模式。在偏斜 XCI 的家族中，我们重新评估了基于 trio 的 ES 并确定了致病性变异和 X 染色体上的缺失。采用连锁分析和 RT-PCR 进一步研究失活的 X 染色体等位基因，采用 Xdrop 长 DNA 技术确定染色体缺失边界。我们发现 16/186 （8.6%） NDD 男性的母亲和 12/90 （13.3%） NDD 女性的 XCI （>90%） 偏斜，远高于正常人群的预期 XCI 发生率 （3.6%，OR = 4.10;OR = 2.51）。通过重新分析 ES 和临床数据，我们解决了 7/28 例 （25%） 的偏斜 XCI，鉴定了 KDM5C 、 PDZD4 、 PHF6 、 TAF1 、 OTUD5 和 ZMYM3 中的变异，以及 ATRX 中的缺失。我们得出结论，XCI 分析是一种简单的检测方法，针对可以从 X 连锁变异的重新评估中受益的患者亚组，从而提高 NDD 患者的诊断率并识别新的 X 连锁疾病。