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首页> 外文学位 >Novel mechanisms of Microphthalmia, Anophthalmia and Coloboma.
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Novel mechanisms of Microphthalmia, Anophthalmia and Coloboma.

机译：小眼，无眼和结肠炎的新机制。

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摘要

Microphthalmia, anophthalmia and coloboma (MAC) form a spectrum of potentially blinding ocular anomalies with a combined prevalence of approximately 1.2 per 10,000 live births. Defined as a reduction in the size of the globe (microphthalmia), absence of the globe (anophthalimia) or failure of the optic fissure to close (coloboma), the MAC spectrum is a highly heterogeneous congenital ocular malformation with over 80 currently known genes and 40-60% of cases still unexplained. In this study, we aimed to use whole exome sequencing (WES) to identify novel factors involved in the MAC spectrum and further analyze their pathogenicity and developmental role using zebrafish as a model.;The second aim was to discover novel factors involved in the MAC spectrum and further evaluate them using zebrafish; this work resulted in two additional publications. Using WES data from the 27 probands negative for variants in known genes, we identified two novel factors associated with MAC. Trio analysis identified a de novo frameshift EFTUD2 variant in a patient with microphthalmia, coloboma, retinal dystrophy, microcephaly and craniofacial defects. Dominant mutations in EFTUD2 cause Mandibulofacial Dysostosis with variable mild ocular features, however MAC has not previously been reported. TALENs were utilized to knockout eftud2. Homozygous mutants displayed reduced head size, smaller eyes, curved body, massive cell death and early embryonic lethality. This is the first patient with a variant in EFTUD2 to be associated with severe ocular phenotypes.;WES of two first cousins affected with coloboma, microcornea, cataracts and skeletal dysplasia revealed a shared heterozygous MAB21L2 variant, p.(Arg51Gly). The variant completely co-segregated with disease phenotype in affected family. TALEN mediated genome editing created a frameshift loss-of-function allele, mab21l2Q48Sfs*5, and an inframe two amino acid deletion, mab21l2R51_F52del, encompassing the orthologous Arg51. Homozygous embryos for mab21l2Q48Sfs*5 presented with severe lens and retinal defects while homozygous mab21l2R51_F52del mutants displayed a milder lens phenotype and more severe coloboma. Both lines demonstrated optic vesicle invagination defects, increased cell death/abnormal proliferation and altered expression of several ocular markers. Importantly, mRNA rescue assays demonstrated that wild type MAB21L2 mRNA was able to rescue the mab21l2Q48Sfs*5 phenotype while mRNA encoding for the p.(Arg51Gly) variant was not able to do the same. These findings support the identification of MAB21L2 as a novel factor involved in human coloboma.;The third aim of this study was to further explore the role of the MAB21L/mab21l family during development. Screening of MAB21L1 in patients with MAC revealed a heterozygous variant of uncertain significance, p.(Arg62Cys). A frameshift loss-offunction allele, mab21l1K36Rfs*7, was created using TALENs to further explore MAB21L1/mab21l1's role in development. Homozygous mab21l1K36Rfs*7 embryos lacked an obvious embryonic phenotype, but homozygous adults displayed ocular coloboma. These findings suggest an independent and important function for MAB21L1/mab21l1 in ocular development.;The first aim of this study was to screen MAC patients for pathogenic variants in known ocular genes. We examined the WES data of 32 MAC probands, as well as available family members, for variants in known MAC and other ocular genes. We identified likely pathogenic variants in COL4A1, OTX2, PAX6 and NDP. In the case of COL4A1, we were the first group to demonstrate a clear association of this gene with MAC phenotypes. For PAX6, the novel missense variant we identified is the first variant associated with aphakia. These findings were reported in two separate first-author publications.;To further explore previous reports of genetic interactions between Pax6 and Mab21l2, we created double heterozygous zebrafish with the loss-of-function pax6bsa86 allele and either mab21l2Q48Sfs*5 or mab21l2R51_F52del alleles. The double homozygous embryos (mab21l2/pax6b), as well as embryos with both a homozygous mutation (mab21l2 or pax6b) and a heterozygous mutation (pax6b or mab21l2), demonstrated more severe phenotypes than single homozygous embryos for either gene with novel ocular features present in some cases. This indicates that mab21l2 and pax6b are likely involved in overlapping as well as parallel pathways.;Finally, to gain a better understanding of mab21l2 function, we performed microarray analysis to identify transcriptome wide changes in gene expression due to mab21l2 deficiency. Analysis of differentially expressed transcripts identified enrichment of genes associated with cataracts, microphthalmia and other ocular phenotypes. Genes belonging to tbx and vsx families, as well as several genes involved in the retinoic acid signaling pathway, were the most significantly affected.;In conclusion, this study expanded the genetic and phenotypic spectrum of MAC disorders. Most importantly, this project identified a novel human disease gene, MAB21L2, and obtained an initial insight into molecular mechanisms associated with its deficiency. This project additionally generated several genetic models of mab21l1 and mab21l2 deficiency in zebrafish that can now be utilized for further studies of this debilitating visual disorder and development of new therapeutic strategies.
机译：小眼症，无眼症和眼球瘤（MAC）形成了一系列潜在的致盲眼异常，其总患病率约为每10,000个活产儿1.2。 MAC频谱被定义为球状体缩小（小眼症），球状体缺失（无眼球症）或视神经裂闭失败（结肠炎），是一种高度异质的先天性眼畸形，具有80多个目前已知的基因， 40-60％的病例仍无法解释。在这项研究中，我们旨在使用全外显子组测序（WES）来识别MAC谱中涉及的新因素，并以斑马鱼为模型进一步分析其致病性和发育作用。第二个目的是发现MAC中涉及的新因素。光谱并使用斑马鱼进一步评估它们；这项工作导致了另外两本出版物。使用来自已知基因变异阴性的27个先证者的WES数据，我们确定了与MAC相关的两个新因素。三重分析分析确定了患有小眼病，眼球瘤，视网膜营养不良，小头畸形和颅面部缺陷的患者的从头移码EFTUD2变异。 EFTUD2中的显着突变会导致下颌面肌营养不良，其轻度眼部特征会有所变化，但是MAC以前尚未见报道。利用TALENs敲除eftud2。纯合突变体显示出头部缩小，眼睛变小，弯曲的身体，大量细胞死亡和早期胚胎致死率。这是首例出现EFTUD2变异与严重眼表型相关的患者;两个最早患表皮瘤，微角膜，白内障和骨骼发育不良的表兄弟的WES揭示了一个共享的杂合MAB21L2变异p。（Arg51Gly）。该变异与受影响家庭的疾病表型完全共隔离。 TALEN介导的基因组编辑创建了一个移码功能丧失等位基因mab21l2Q48Sfs * 5和一个读框内两个氨基酸缺失mab21l2R51_F52del，涵盖了同源Arg51。 mab21l2Q48Sfs * 5的纯合子胚胎具有严重的晶状体和视网膜缺陷，而纯合的mab21l2R51_F52del突变体表现出较轻的晶状体表型和更严重的结肠癌。两种品系均显示出视神经泡内陷缺陷，细胞死亡增加/异常增殖以及几种眼标记物的表达改变。重要的是，mRNA拯救分析表明野生型MAB21L2 mRNA能够拯救mab21l2Q48Sfs * 5表型，而编码p。（Arg51Gly）变体的mRNA却不能做到这一点。这些发现支持将MAB21L2鉴定为涉及人类淋巴瘤的新因素。本研究的第三个目标是进一步探索MAB21L / mab21l家族在发育过程中的作用。对患有MAC的患者的MAB21L1进行筛查发现，杂合子变异体的意义不确定，p。（Arg62Cys）。使用TALENs创建了移码功能丧失等位基因mab21l1K36Rfs * 7，以进一步探索MAB21L1 / mab21l1在发育中的作用。纯合子mab21l1K36Rfs * 7胚胎缺乏明显的胚胎表型，但纯合子成虫显示眼球状瘤。这些发现表明，MAB21L1 / mab21l1在眼部发育中具有独立且重要的功能。这项研究的首要目的是筛选MAC患者已知眼基因中的致病变异。我们检查了32个MAC先证者的WES数据以及可用的家庭成员，以了解已知MAC和其他眼基因的变体。我们确定了COL4A1，OTX2，PAX6和NDP中可能的致病变异。就COL4A1而言，我们是第一个证明该基因与MAC表型有明确关联的小组。对于PAX6，我们确定的新型错义变异是与无晶状体相关的第一个变异。这些发现在两个单独的第一作者的出版物中进行了报道。为了进一步探索Pax6和Mab21l2之间的遗传相互作用的先前报道，我们创建了功能失常的pax6bsa86等位基因和mab21l2Q48Sfs * 5或mab21l2R51_F52del等位基因的双杂合斑马鱼。双纯合胚胎（mab21l2 / pax6b）以及具有纯合突变（mab21l2或pax6b）和杂合突变（pax6b或mab21l2）的胚胎表现出比单一纯合子胚胎更严重的表型，这两个基因均具有新颖的眼部特征在某些情况下。这表明mab21l2和pax6b可能参与了重叠和平行途径。最后，为了更好地了解mab21l2的功能，我们进行了微阵列分析，以鉴定由于mab21l2缺乏引起的转录组基因表达的宽泛变化。分析差异表达的转录本，发现与白内障，小眼症和其他眼表型有关的基因富集。属于tbx和vsx家族的基因以及视黄酸信号通路中涉及的几个基因受到的影响最大。，这项研究扩大了MAC疾病的遗传和表型谱。最重要的是，该项目鉴定了一种新的人类疾病基因MAB21L2，并初步了解了与之相关的分子机制。该项目还生成了斑马鱼中mab21l1和mab21l2缺乏症的几种遗传模型，现在可以将其用于进一步研究这种使人衰弱的视觉障碍并开发新的治疗策略。

著录项

  • 作者

    Deml, Brett Allen.;

  • 作者单位

    The Medical College of Wisconsin.;

  • 授予单位 The Medical College of Wisconsin.;
  • 学科 Genetics.;Molecular biology.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 271 p.
  • 总页数 271
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 高分子化学（高聚物）;
  • 关键词

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