Characterization of a novel combined immunodeficiency with skeletal dysplasia

 描述（由申请人提供）：免疫性骨发育不良（IOD）是一组以免疫缺陷和骨骼发育不良为特征的疾病。Schimke病（SIOD）和软骨毛发发育不全（CHH）代表了这些疾病的原型形式，但其他几例IOD的分子基础未知已被描述。IOD中的免疫缺陷通常很严重，主要影响T淋巴细胞。在一些CHH患者中报告了Omenn综合征（OS）典型的免疫失调。我们已经确定并研究了两个兄弟姐妹（一男一女）与快速进展和致命的IOD。两名婴儿均表现出T细胞淋巴细胞减少症，CD 8 + T细胞数量显著减少，缺乏对有丝分裂原的增殖，低丙种球蛋白血症，血清IgE和嗜酸性粒细胞增多症增加。哥哥有OS的特点，红皮病和扩增的寡克隆和活化的CD 4 + T细胞。SMARCAL 1和RMRP基因中未发现突变，分别负责SIOD和CHH。全外显子组测序（WES）显示，两个兄弟姐妹是纯合子，两个父母是杂合子，一个新的和激烈的错义突变（R339 W）影响一个高度保守的外生骨蛋白样3（EXTL 3），一个成员的外生骨蛋白（EXT）家族的糖基转移酶参与硫酸乙酰肝素（HS）的生物合成的残基。HS是修饰细胞表面的硫酸化糖胺聚糖和基质蛋白聚糖。HS蛋白聚糖调节在骨骼发育和T细胞分化中起关键作用的形态发生素的活性。我们的总体假设是，EXTL 3对胸腺发育至关重要，人类EXTL 3突变导致一种新形式的IOD。为了支持我们的假设，我们的初步数据表明，EXTL 3表达的人胸腺细胞，胸腺上皮细胞（TEC），和外周T细胞。此外，在斑马鱼中Extl 3的突变导致肢芽发育缺陷和胸腺T细胞发育受损。为了检验我们的假设，并调查与EXTL 3突变相关的免疫缺陷的细胞基础，我们将产生和表征小鼠模型与神经嵴间充质细胞，TEC，或胸腺细胞中的Extl 3基因的组织特异性破坏。所有这些细胞类型都会产生HS，并且在胸腺器官发生和T细胞发育中发挥重要作用。同时，为了证明在受影响的兄弟姐妹中鉴定的EXTL 3 p.R339W突变的致病作用，我们将使用患者来源的诱导多能干细胞（iPSC）进行疾病建模。我们将使用CRISPR/Cas9技术来纠正患者来源的iPSC中的EXTL 3 p.R339W突变。为了确定EXTL 3突变的细胞自主效应，我们将突变和基因校正的iPSC分化为TEC和T细胞，并且我们将在相同的实验条件下研究HS的生物合成。我们预计，这项研究将提供新的重要见解的生物学作用的HSPGs在免疫系统的发展和功能，并将有助于确定最佳的治疗方法，以纠正这种情况下的免疫缺陷。

 DESCRIPTION (provided by applicant): Immuno-osseous dysplasias (IOD) are a group of disorders characterized by immune deficiency and skeletal dysplasia. Schimke's disease (SIOD) and cartilage hair hypoplasia (CHH) represent the prototypic forms of these disorders, but several other cases of IOD with unknown molecular basis have been described. The immunodeficiency seen in IOD is often severe, and affects predominantly T lymphocytes. Immune dysregulation typical of Omenn syndrome (OS) has been reported in some patients with CHH. We have identified and studied two siblings (a male and a female) with rapidly progressive and fatal IOD. Both infants manifested T cell lymphopenia with markedly reduced number of CD8+ T cells, lack of proliferation to mitogens, hypogammaglobulinemia, increased serum IgE and eosinophilia. The elder sibling had features of OS, with erythroderma and expansion of oligoclonal and activated CD4+ T cells. No mutations were identified in the SMARCAL1 and RMRP genes, responsible for SIOD and CHH, respectively. Whole exome sequencing (WES) revealed that both siblings were homozygous, and both parents heterozygous, for a novel and drastic missense mutation (R339W) affecting a highly conserved residue of Exostosin-like 3 (EXTL3), a member of the exostosin (EXT) family of glycosyltransferases involved in heparan sulfate (HS) biosynthesis. HS are sulfated glycosaminoglycans that decorate cell surface and matrix proteoglycans. HS proteoglycans modulate the activity of morphogens that play a key role in skeletal development and T cell differentiation. Our overall hypothesis is that EXTL3 is critical for thymic development and that EXTL3 mutations in humans cause a novel form of IOD. In support of our hypothesis, our preliminary data demonstrate that EXTL3 is expressed by human thymocytes, thymic epithelial cells (TECs), and peripheral T cells. Moreover, mutation of Extl3 in zebrafish causes defects of limb bud development, and impaired thymic T cell development. To test our hypothesis, and to investigate the cellular basis of the immunodeficiency associated with EXTL3 mutations, we will generate and characterize mouse models with tissue-specific disruption of the Extl3 gene in neural crest mesenchyme cells, TECs, or thymocytes. All of these cell types produce HS and are critically involved in thymus organogenesis and T cell development. In parallel, in order to demonstrate the disease-causing effect of the EXTL3 p.R339W mutation identified in the affected siblings, we will perform disease modeling using patient-derived induced pluripotent stem cells (iPSCs). We will use the CRISPR/Cas9 technology to correct the EXTL3 p.R339W mutation in patient-derived iPSCs. In order to define the cell autonomous effect of the EXTL3 mutation, we will differentiate both mutant and gene-corrected iPSCs to TECs and T cells, and we will study HS biosynthesis under the same experimental conditions. We anticipate that this study will provide novel important insights on the biological role of HSPGs in immune system development and function, and will help define the optimal therapeutic approach to correct the immunodeficiency of this condition.