Impaired B-cell function in ERCC2 deficiency. Impaired B-cell function in ERCC2 deficiency

Trichothiodystrophy-1 (TTD1) is an autosomal-recessive disease and caused by mutations in ERCC2, a gene coding for a subunit of the TFIIH transcription and nucleotide-excision repair (NER) factor. In almost half of these patients infectious susceptibility has been reported but the underlying molecular mechanism leading to immunodeficiency is largely unknown. The aim of this study was to perform extended molecular and immunological phenotyping in patients suffering from TTD1. Cellular immune phenotype was investigated using multicolour flow cytometry. DNA repair efficiency was evaluated in UV-irradiation assays. Furthermore, early BCR activation events and proliferation of TTD1 lymphocytes following DNA damage induction was tested. In addition, we performed differential gene expression analysis in peripheral lymphocytes of TTD1 patients. We investigated three unrelated TTD1 patients who presented with recurrent infections early in life of whom two harboured novel ERCC2 mutations and the third patient is a carrier of previously described pathogenic ERCC2 mutations. Hypogammaglobulinemia and decreased antibody responses following vaccination were found. TTD1 B-cells showed accumulation of g-H2AX levels, decreased proliferation activity and reduced cell viability following UV-irradiation. mRNA sequencing analysis revealed significantly downregulated genes needed for B-cell development and activation. Analysis of B-cell subpopulations showed low numbers of naïve and transitional B-cells in TTD1 patients, indicating abnormal B-cell differentiation in vivo. In summary, our analyses confirmed the pathogenicity of novel ERCC2 mutations and show that ERCC2 deficiency is associated with antibody deficiency most likely due to altered B-cell differentiation resulting from impaired B-cell activation and activation-induced gene transcription. Overall design: To investigate the differential gene expression in TTD1 patients, we isolated peripheral blood mononuclear cells from 3 unrelated ERCC2 deficient patients as well as 5 healthy controls. Then, RNA was isolated from PBMCs, and total RNA served as input for mRNA sequencing analysis. Bioinformatic analysis following mRNA sequencing should reveal whether impaired or dysregulated transcription in PBMCs from TTD1 patients contributes to impaired antibody response found in these patients.

毛发硫营养不良症1型（Trichothiodystrophy-1, TTD1）是一种常染色体隐性遗传病，由ERCC2基因突变所致；该基因编码转录因子IIH（TFIIH）与核苷酸切除修复（nucleotide-excision repair, NER）因子的一个亚基。目前已有报道显示，近半数此类患者存在感染易感性升高的情况，但导致免疫缺陷的潜在分子机制仍未明确。本研究旨在对罹患TTD1的患者开展扩展分子与免疫学表型分析。研究采用多色流式细胞术检测细胞免疫表型，通过紫外线辐照实验（UV-irradiation assays）评估DNA修复效率，并进一步检测DNA损伤诱导后TTD1淋巴细胞的早期B细胞受体（B-cell receptor, BCR）活化事件与增殖情况。此外，本研究还对TTD1患者的外周淋巴细胞进行了差异基因表达分析。本研究纳入3名无亲缘关系的TTD1患者，均在幼年早期出现复发性感染；其中2名患者携带新型ERCC2突变，另1名患者为既往报道的致病性ERCC2突变携带者。研究发现，患者存在低丙种球蛋白血症（hypogammaglobulinemia），且疫苗接种后抗体应答减弱。TTD1患者的B细胞经紫外线辐照后，g-H2AX水平蓄积、增殖活性降低且细胞存活率下降。mRNA测序分析显示，与B细胞发育及活化相关的基因显著下调。B细胞亚群分析结果表明，TTD1患者体内初始B细胞与过渡型B细胞数量减少，提示体内B细胞分化异常。综上，本研究证实了新型ERCC2突变的致病性，并揭示ERCC2缺陷与抗体缺陷相关，其潜在机制可能为B细胞活化受损及活化诱导的基因转录异常导致B细胞分化改变。总体实验设计：为探究TTD1患者的差异基因表达情况，本研究从3名无亲缘关系的ERCC2缺陷患者及5名健康对照者体内分离外周血单个核细胞（peripheral blood mononuclear cells, PBMCs），随后从PBMCs中提取总RNA，以总RNA作为mRNA测序的起始模板。mRNA测序后的生物信息学分析旨在明确TTD1患者PBMCs中受损或失调的转录过程是否与该类患者所观察到的抗体应答受损相关。