背景:Christianson综合征(CS)是由SLC9A6的突变引起的,其特征是整体发育迟缓,癫痫,运动过度,共济失调,小头畸形,和行为障碍。然而,这些SLC9A6突变导致人类CS的分子机制尚不完全清楚,并且没有客观的方法来确定单个SLC9A6变体的致病性。
方法:对两名怀疑CS的个体进行了基于Trio的全外显子组测序(WES)。qRT-PCR,蛋白质印迹分析,filipin染色,溶酶体酶分析,和电子显微镜检查,使用从两名患者中建立的EBV-LCL,被执行了。
结果:基于三重奏的WES确定了半合子SLC9A6c.1560dupT,p.T521Yfs*23在先证者1中的变体和半合子SLC9A6c.608delA,p.H203Lfs*10在先证者2中的变体。两个孩子都表现出与CS相关的典型表型。来自两名患者的EBV-LCL中的表达分析显示mRNA水平显着降低,并且没有检测到正常的NHE6蛋白。EBV-LCL在患者1中显示出未酯化胆固醇的统计学显着增加,但在患者2中用filipin染色时仅显示出无显着增加。溶酶体酶的活性(β-己糖胺酶A,β-己糖胺酶A+B,β-半乳糖苷酶,半乳糖苷酶,EBV-LCL的芳基硫酸酯酶A)在两名患者和六名对照之间没有显着差异。重要的是,通过电子显微镜,我们检测到层状膜结构的积累,变形的线粒体,和患者EBV-LCLs中的脂滴。
结论:我们患者的SLC9A6p.T521Yfs*23和p.H203Lfs*10变体导致NHE6丢失。线粒体和脂质代谢的改变可能在CS的发病机制中起作用。此外,filipin染色与患者淋巴母细胞的电子显微镜检查相结合,可以作为CS的有用补充诊断方法。
Christianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized by global developmental delay, epilepsy, hyperkinesis, ataxia, microcephaly, and behavioral disorder. However, the molecular mechanism by which these SLC9A6 mutations cause CS in humans is not entirely understood, and there is no objective method to determine the pathogenicity of single SLC9A6 variants.
Trio-based whole exome sequencing (WES) was carried out on two individuals with suspicion of CS. qRT-PCR, western blot analysis, filipin staining, lysosomal enzymatic assays, and electron microscopy examination, using EBV-LCLs established from the two patients, were performed.
Trio-based WES identified a hemizygous SLC9A6 c.1560dupT, p.T521Yfs*23 variant in proband 1 and a hemizygous SLC9A6 c.608delA, p.H203Lfs*10 variant in proband 2. Both children exhibited typical phenotypes associated with CS. Expression analysis in EBV-LCLs derived from the two patients showed a significant decrease in mRNA levels and no detectable normal
NHE6 protein. EBV-LCLs showed a statistically significant increase in unesterified cholesterol in patient 1, but only non-significant increase in patient 2 when stained with filipin. Activities of lysosomal enzymes (β-hexosaminidase A, β-hexosaminidase A + B, β-galactosidase, galactocerebrosidase, arylsulfatase A) of EBV-LCLs did not significantly differ between the two patients and six controls. Importantly, by electron microscopy we detected an accumulation of lamellated membrane structures, deformed mitochondria, and lipid droplets in the patients\' EBV-LCLs.
The SLC9A6 p.T521Yfs*23 and p.H203Lfs*10 variants in our patients result in loss of
NHE6. Alterations of mitochondria and lipid metabolism may play a role in the pathogenesis of CS. Moreover, the combination of filipin staining with electron microscopy examination of patient lymphoblastoid cells can serve as a useful complementary diagnostic method for CS.