Zhi Xiufang, Ai Qi, Sheng Wenchao, Yu Yuping, Shu Jianbo, Yu Changshun, Yu Xiaoli, Li Dong, Cai Chunquan
Graduate College of Tianjin Medical University, Tianjin, China.
Tianjin Children's Hospital (Children's Hospital of Tianjin University), Tianjin, China.
Front Genet. 2022 Mar 31;13:852764. doi: 10.3389/fgene.2022.852764. eCollection 2022.
Menkes disease (MD) is a rare X-linked connective tissue disorder of copper metabolism caused by pathogenic variant(s) in gene. The aim of the present study is to determine the clinical characteristics and molecular basis of one patient with MD. One 10-month-old Chinese boy who met the clinical manifestations of MD was enrolled in this study. Whole genome sequencing (WGS) was performed in the patient in order to identify the variant(s), followed by Sanger sequencing. RNA sequencing (RNA-seq) from whole blood was subsequently applied to assess the effect of variant on transcription levels, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for further validation. In addition, X chromosome inactivation (XCI) status of the patient's mother at the DNA level was measured by capillary electrophoresis. The patient suffered from intermittent convulsions for more than 6 months, with psychomoto retardation and neurodegenerations. The patient also had curly hair, hypopigmented skin, cutis laxa, decreased muscle strength and hypotonia. MRI showed the intracranial arteries were tortuous with some "spiral" changes. The patient's serum ceruloplasmin level was low. WGS revealed one novel hemizygous variant, c.2627-501C > T (NM_000,052.7), located in the deep intronic sequence of gene. Sanger sequencing confirmed that the variant was inherited from his mother. RNA-seq confirmed the variant itself, and identified a pseudo-exon inserted between exons 12 and 13 in mRNA of . The sequencing results of RT-PCR from the patient confirmed this finding, while neither of his parents detected aberrant splicing. The Capillary electrophoresis results showed that the patient's mother had a skewed XCI. Our finding of the variant enlarges the variant spectrum in the gene. This is a novel deep intronic variant which leads to the activation of a pseudo-exons in the gene, and it demonstrates the usefulness of WGS combined with RNA-seq, in terms of revealing disease-causing variants in non-coding regions. Furthermore, the fact that the deep intronic variants cause disease by the activation of pseudo-exon inclusion indicates that in MD this might be an important mechanism.
门克斯病（MD）是一种罕见的X连锁铜代谢结缔组织疾病，由该基因中的致病变异引起。本研究的目的是确定一名MD患者的临床特征和分子基础。一名符合MD临床表现的10个月大中国男孩被纳入本研究。对该患者进行全基因组测序（WGS）以鉴定变异，随后进行桑格测序。随后应用全血RNA测序（RNA-seq）来评估变异对转录水平的影响，并进行逆转录聚合酶链反应（RT-PCR）以进一步验证。此外，通过毛细管电泳检测患者母亲在DNA水平的X染色体失活（XCI）状态。该患者间歇性抽搐超过6个月，伴有精神运动发育迟缓及神经退行性变。患者还出现卷发、皮肤色素减退、皮肤松弛、肌力下降和肌张力减退。磁共振成像（MRI）显示颅内动脉迂曲，有一些“螺旋”样改变。患者血清铜蓝蛋白水平较低。WGS揭示了一个新的半合子变异，c.2627-501C>T（NM_000,052.7），位于该基因的内含子深部序列。桑格测序证实该变异遗传自其母亲。RNA-seq证实了该变异本身，并在该基因的mRNA中鉴定出一个插入在第12和13外显子之间的假外显子。该患者RT-PCR的测序结果证实了这一发现，而其父母均未检测到异常剪接。毛细管电泳结果显示患者母亲存在XCI偏斜。我们对该变异的发现扩大了该基因的变异谱。这是一个新的内含子深部变异，导致该基因中一个假外显子的激活，并且它证明了WGS与RNA-seq相结合在揭示非编码区致病变异方面的有用性。此外，内含子深部变异通过激活假外显子的包含而致病这一事实表明，在MD中这可能是一个重要机制。
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