Wang Xiao-Ming, Yik Wing Yan, Zhang Peilin, Lu Wange, Dranchak Patricia K, Shibata Darryl, Steinberg Steven J, Hacia Joseph G
Stem Cell Res Ther. 2012 Oct 4;3(5):39. doi: 10.1186/scrt130.
X-linked adrenoleukodystrophy (X-ALD) is a complex disorder with variable expressivity that affects the nervous, adrenocortical and male reproductive systems. Although ABCD1 mutations are known to provide the genetic basis for X-ALD, its pathogenesis is not fully elucidated. While elevated very long chain fatty acid (VLCFA) levels in blood and reduced VLCFA catabolic activity in cultured fibroblasts are biomarkers used to identify ABCD1 mutation carriers, the roles peroxisomal lipid metabolism play in disease etiology are unknown.
Primary skin fibroblasts from two male patients with the childhood cerebral form of the disease (CCALD) caused by ABCD1 frameshift or missense mutations and three healthy donors were transduced with retroviral vectors expressing the OCT4, SOX2, KLF4 and c-MYC factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, DNA copy number variation, and genotyping analysis and tested for pluripotency through in vitro differentiation and teratoma formation. Saturated VLCFA (sVLCFA) and plasmalogen levels in primary fibroblasts and iPSCs from healthy donors as well as CCALD patients were determined through mass spectroscopy.
Skin fibroblasts from CCALD patients and healthy donors were reprogrammed into validated iPSCs. Unlike fibroblasts, CCALD patient iPSCs show differentially expressed genes (DEGs) relevant to both peroxisome abundance and neuroinflammation. Also, in contrast to fibroblasts, iPSCs from patients showed no significant difference in sVLCFA levels relative to those from controls. In all cell types, the plasmalogen levels tested did not correlate with ABCD1 mutation status.
Normal ABCD1 gene function is not required for reprogramming skin fibroblasts into iPSCs or maintaining pluripotency. Relative to DEGs found in fibroblasts, DEGs uncovered in comparisons of CCALD patient and control iPSCs are more consistent with major hypotheses regarding disease pathogenesis. These DEGs were independent of differences in sVLCFA levels, which did not vary according to ABCD1 mutation status. The highlighted genes provide new leads for pathogenic mechanisms that can be explored in animal models and human tissue specimens. We suggest that these iPSC resources will have applications that include assisting efforts to identify genetic and environmental modifiers and screening for therapeutic interventions tailored towards affected cell populations and patient genotypes.
X连锁肾上腺脑白质营养不良（X-ALD）是一种具有可变表达性的复杂疾病，会影响神经、肾上腺皮质和男性生殖系统。虽然已知ABCD1突变是X-ALD的遗传基础，但其发病机制尚未完全阐明。血液中极长链脂肪酸（VLCFA）水平升高以及培养的成纤维细胞中VLCFA分解代谢活性降低是用于识别ABCD1突变携带者的生物标志物，但过氧化物酶体脂质代谢在疾病病因中的作用尚不清楚。
用表达OCT4、SOX2、KLF4和c-MYC因子的逆转录病毒载体转导两名患有由ABCD1移码或错义突变引起的儿童脑型疾病（CCALD）的男性患者和三名健康供体的原代皮肤成纤维细胞。对候选诱导多能干细胞（iPSC）进行全基因组表达、DNA甲基化、DNA拷贝数变异和基因分型分析，并通过体外分化和畸胎瘤形成测试其多能性。通过质谱法测定健康供体以及CCALD患者的原代成纤维细胞和iPSC中的饱和VLCFA（sVLCFA）和缩醛磷脂水平。
CCALD患者和健康供体的皮肤成纤维细胞被重编程为经过验证的iPSC。与成纤维细胞不同，CCALD患者的iPSC显示出与过氧化物酶体丰度和神经炎症相关的差异表达基因（DEG）。此外，与成纤维细胞相比，患者的iPSC在sVLCFA水平上与对照组没有显著差异。在所有细胞类型中，测试的缩醛磷脂水平与ABCD1突变状态无关。
将皮肤成纤维细胞重编程为iPSC或维持多能性不需要正常的ABCD1基因功能。相对于在成纤维细胞中发现的DEG，在CCALD患者和对照iPSC的比较中发现的DEG与关于疾病发病机制的主要假设更一致。这些DEG与sVLCFA水平的差异无关，sVLCFA水平不会因ABCD1突变状态而变化。突出显示的基因提供了可在动物模型和人体组织标本中探索的致病机制的新线索。我们建议这些iPSC资源将有多种应用，包括协助识别遗传和环境修饰因子的工作，以及筛选针对受影响细胞群体和患者基因型的治疗干预措施。
