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首页> 外文学位 >Studies of factors involved in PITX2 regulation and their role in anterior segment dysgenesis phenotypes.
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Studies of factors involved in PITX2 regulation and their role in anterior segment dysgenesis phenotypes.

机译：研究PITX2调控相关因素及其在前节发育不全表型中的作用。

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摘要

Axenfeld-Rieger syndrome (ARS) is characterized by malformations of the anterior segment of the eye, craniofacial and dental defects, and redundant periumbilical skin. Mutations in PITX2 are associated with ARS; however, mutations in PITX2 or other known genes explain only ∼40% of ARS cases. Identification of cis-regulatory elements of PITX2 can aid in elucidating the mechanisms of disease. Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Conserved non-coding elements surrounding PITX2/ pitx2 were identified and examined through transgenic analysis in zebrafish. Thirteen conserved non-coding sequences positioned within a gene desert as far as 1.1-Mb upstream of the human PITX2 gene were identified. One region, CE4, located approximately 111-kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, consistent with the sites affected in ARS. Screening of ARS patients identified two patients with genomic deletions located 50-kb and 106-108-kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4-CE13. These data support the significance of distant upstream regulatory elements in normal gene function and offer a possible mechanism for ARS in patients with balanced translocations and deletions involving the upstream PITX2 region. The CE4 sequence was examined for putative interacting proteins, and multiple forkhead protein consensus binding sites were identified within or near CE4. FOXD3 encodes a forkhead-domain transcription factor that is expressed in precursor and early migratory neural crest cells which contribute to the developing anterior segment structures of the eye. A role for FOXD3 in human eye disease has not been reported. In zebrafish, foxd3 is expressed in the neural-crest derived periocular mesenchyme, as well as in the hindbrain and pharyngeal arches. A novel foxd3-deficient zebrafish line harboring a 5.7-kb viral insertion incorporated into the forkhead DNA-binding domain and predicted to encode a mutant protein containing 33% of normal foxd3 sequence (including only 30% of the forkhead domain) and 12 erroneous amino acids encoded by the viral sequence was characterized. Homozygous mutant fish display craniofacial and cardiac defects with embryonic lethality by 7-10 days post-fertilization (dpf), while heterozygous embryos appear normal. While no major structural changes in the eye were observed in up to 5-dpf mutant embryos, a reduction in expression of several genes known to be important for anterior segment development (including pitx2 ) was observed. FOXD3 was next evaluated for its contribution to human ocular disease. 310 probands with developmental ocular conditions were screened for variation in FOXD3. Six nonsynonymous FOXD3 variants were identified. Four of these changes, c.47C>T (p.Thr16Met), c.359C>T (p.Pro120Leu), c.517A>C (p.Asn173His), and c.818_829dup (p.Arg273_Gly276dup), affected conserved regions and were observed primarily in probands with aniridia or Peters anomaly; out of these four variants, p.Arg273_Gly276dup was not detected in control populations and p.Pro120Leu and p.Asn173His were statistically enriched in cases with aniridia or Peters anomaly. The p.Asn173His missense mutation affects a 100% conserved residue among vertebrate orthologs located within the forkhead domain and is predicted to be involved in protein-protein interactions rather than direct contact with DNA. In summary, these data suggest that a complex distant regulatory matrix located upstream of PITX2 plays an essential role in gene activity and its deletion provides a novel mechanism for ARS. FOXD3/foxd3 may play a role in normal eye development and contribute to human disease. Identification of co-factors that may modulate FOXD3 interactions and further analysis of foxd3-deficient zebrafish lines are necessary to better understand the contribution of FOXD3/foxd3 to ocular phenotypes, as well as determine relationships between FOXD3 and PITX2..
机译：Axenfeld-Rieger综合征（ARS）的特征是眼前节畸形，颅面和牙齿缺损以及多余的脐周皮肤。 PITX2中的突变与ARS相关；然而，PITX2或其他已知基因的突变仅解释了约40％的ARS病例。 PITX2的顺式调节元件的鉴定可以帮助阐明疾病的机制。斑马鱼pitx2在眼和颅面发育过程中表现出保守的表达。通过转基因分析在斑马鱼中鉴定并检测了PITX2 / pitx2周围的保守非编码元件。鉴定了位于人类PITX2基因上游1.1-Mb的基因沙漠中的13个保守非编码序列。位于PITX2上游约111 kb的一个区域CE4指向一种复杂的模式，包括在发育中的眼睛和颅面区域中的表达，与ARS中受影响的部位一致。 ARS患者的筛查确定了两名基因缺失的患者，位于PITX2基因上游50-kb和106-108-kb，保留了PITX2完整，同时去除了调控元件CE4-CE13。这些数据支持正常基因功能中远距离上游调控元件的重要性，并为涉及上游PITX2区的平衡易位和缺失的患者提供了ARS的可能机制。检查CE4序列是否存在相互作用蛋白，并在CE4内或附近鉴定出多个叉头蛋白共有结合位点。 FOXD3编码在前体和早期迁徙的神经rest细胞中表达的叉头结构域转录因子，其有助于眼睛的前节结构的发育。尚未报道FOXD3在人眼疾病中的作用。在斑马鱼中，foxd3在源自神经c的眼周间充质以及后脑和咽弓中表达。一种新型的foxd3缺陷型斑马鱼品系，其向叉头DNA结合域掺入了5.7kb的病毒插入，并预测其编码的突变蛋白含有33％的正常foxd3序列（仅包含叉头域的30％）和12个错误的氨基表征了由病毒序列编码的酸。纯合突变鱼在受精后（dpf）到7-10天时显示出颅面和心脏缺陷，具有胚胎致死性，而杂合子胚胎则正常。虽然在最多5-dpf突变的胚胎中未观察到眼睛的主要结构变化，但观察到已知对前节发育非常重要的几种基因（包括pitx2）的表达减少。接下来评估FOXD3对人眼疾病的贡献。筛选了310名具有发育性眼病的先证者的FOXD3变异。鉴定出六个非同义的FOXD3变体。这些变化中的四个变化（c.47C> T（p.Thr16Met），c.359C> T（p.Pro120Leu），c.517A> C（p.Asn173His）和c.818_829dup（p.Arg273_Gly276dup）受保护）区域，主要在患有虹膜异常或彼得斯异常的先证者中观察到；在这四个变体中，在对照人群中未检测到p.Arg273_Gly276dup，而在患有虹膜异位症或Peters异常的病例中，p.Pro120Leu和p.Asn173His在统计学上富集。 p.Asn173His的错义突变会影响位于叉头结构域内的脊椎动物直系同源基因中100％的保守残基，并被预测会参与蛋白质间相互作用，而不是直接与DNA接触。总之，这些数据表明，位于PITX2上游的复杂的远距离调控基质在基因活性中起着至关重要的作用，其缺失为ARS提供了一种新的机制。 FOXD3 / foxd3可能在正常的眼睛发育中起作用，并导致人类疾病。为了更好地了解FOXD3 / foxd3对眼表型的贡献，以及确定FOXD3与PITX2之间的关系，必须鉴定可能调节FOXD3相互作用的辅因子并进一步分析foxd3缺陷型斑马鱼系。

著录项

  • 作者

    Volkmann Kloss, Bethany Anne.;

  • 作者单位

    The Medical College of Wisconsin.;

  • 授予单位 The Medical College of Wisconsin.;
  • 学科 Biology Molecular.;Biology Cell.;Biology Genetics.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 196 p.
  • 总页数 196
  • 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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