Characterization of a novel combined immunodeficiency with skeletal dysplasia

一种新型联合免疫缺陷伴骨骼发育不良的特征

• 批准号: 8995190
• 负责人: Luigi Daniele Notarangelo
• 金额: $ 22.13万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8995190
• 关键词: Affect; Architecture; Autoantigens; Autologous; Biological; Branchial arch structure; CD4 Positive T Lymphocytes; CD8B1 gene; CRISPR/Cas technology; Cartilage; Cell surface; Cells; Data; Defect; Development; Disease; Disease model; Dysplasia; Elderly; Eosinophilia; Exfoliative Dermatitis; FOXP3 gene; Family; Female; Gene Mutation; Generations; Genes; Hair; Health; Hematopoietic Stem Cell Transplantation; Heparan Sulfate Biosynthesis; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human; IgE; Immune; Immune system; Immunity; Immunologic Deficiency Syndromes; In Vitro; Infant; Infection; Lead; Limb Bud; Link; Lymphatic Diseases; Lymphopenia; Mesenchyme; Missense Mutation; Mitogens; Molecular; Mus; Mutation; Neural Crest; Organogenesis; Parents; Patients; Pectoral; Peripheral; Phenotype; Play; Proteoglycan; Recurrence; Regulatory T-Lymphocyte; Reporting; Role; Serum; Severe Combined Immunodeficiency; Siblings; Skeletal Development; Syndrome; System; Systems Development; T cell differentiation; T-Cell Development; T-Lymphocyte; Technology; Testing; Therapeutic; Thymic epithelial cell; Thymus Gland; Tissues; Zebrafish; base; cell type; congenital immunodeficiency; disease phenotype; exome sequencing; gene correction; glycosyltransferase; induced pluripotent stem cell; insight; male; member; morphogens; mouse model; mutant; novel; polysulfated glycosaminoglycan; skeletal; skeletal dysplasia; thymocyte; thymus transplantation; transcription factor

 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.

 描述(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)和软骨毛发发育不良(CHH)是这些疾病的典型形式，但也描述了其他几个分子基础未知的IOD病例。IOD的免疫缺陷通常是严重的，主要影响T淋巴细胞。一些CHH患者出现典型的Omenn综合征(OS)免疫功能紊乱。我们已经确认和研究了两个兄弟姐妹(一男一女)，他们患有快速进展和致命的IOD。两例患儿均表现为T细胞淋巴细胞减少，CD8+T细胞数量明显减少，对丝裂原缺乏增殖，低丙种球蛋白血症，血清IgE升高，嗜酸性粒细胞增多。姐姐有OS的特征，伴有红皮病，并有寡克隆和活化的CD4+T细胞的增殖。未发现SMARCAL1和RMRP基因突变，这两个基因分别与SIOD和CHH有关。全外显子组测序(WES)显示，两个兄弟姐妹都是纯合子，父母都是杂合子，一个新的严重错义突变(R339W)影响了参与硫酸乙酰肝素(HS)生物合成的Exostosin-like 3(EXTL3)高度保守的残基，Exostosin-like 3(EXTL3)家族的成员。HS是修饰细胞表面和基质蛋白多糖的硫酸化糖胺多聚糖。HS蛋白多糖调节在骨骼发育和T细胞分化中起关键作用的形态原的活性。我们的总体假设是，EXTL3对胸腺发育至关重要，人类的EXTL3突变会导致一种新型的IOD。为了支持我们的假设，我们的初步数据表明，EXTL3在人类胸腺细胞、胸腺上皮细胞(TECs)和外周T细胞中表达。此外，斑马鱼Extl3基因的突变会导致肢芽发育缺陷，并损害胸腺T细胞的发育。为了验证我们的假设，并研究与EXTL3突变相关的免疫缺陷的细胞学基础，我们将建立和表征组织特异性破坏神经脊间充质细胞、TECs或胸腺细胞中Extl3基因的小鼠模型。所有这些细胞类型都会产生HS，并在胸腺器官发生和T细胞发育中起关键作用。同时，为了证明在受影响同胞中发现的EXTL3p.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(IPSCs)进行疾病建模。我们将使用CRISPR/Cas9技术来纠正患者来源的IPSCs中的EXTL3p.R339W突变。为了明确EXTL3突变的细胞自主效应，我们将突变和基因校正的IPSCs分化为TECs和T细胞，并在相同的实验条件下研究HS的生物合成。我们期待这项研究将为HSPGs在免疫系统发育和功能中的生物学作用提供新的重要见解，并将有助于确定纠正这种疾病的免疫缺陷的最佳治疗方法。