Gene Discovery in Human Anopthalmia/Micropthalmia

人类无眼症/小眼症的基因发现

• 批准号: 8364784
• 负责人: ANNE M. SLAVOTINEK
• 金额: $ 23.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364784
• 关键词: Affect; Anophthalmos; Autistic Disorder; Biological; Candidate Disease Gene; Child; Choanal Atresia; Choroid; Clinical; Code; Coloboma; Complex; Computer Simulation; Congenital Abnormality; Counseling; Data; Databases; Diagnosis; Diagnostic; Diaphragmatic Hernia; Dideoxy Chain Termination DNA Sequencing; Disease; Embryo; Etiology; Eye; Eye Development; Failure; Family; Future; Gene Expression; Gene Frequency; Gene Mutation; Genes; Genetic; Genetic Research; Genomics; Grant; Health; Human; In Situ Hybridization; Individual; Inheritance Patterns; Inherited; Injection of therapeutic agent; Intellectual functioning disability; Knowledge; Life Style; Live Birth; Medical; Messenger RNA; Methodology; Microphthalmos; Molecular; Mus; Mutation; Mutation Detection; Orthologous Gene; Parents; Pathogenesis; Patient Care; Patients; Phenotype; Pilot Projects; Proteins; Reading; Research; Severities; Single Nucleotide Polymorphism; Syndrome; Systems Biology; Technology; United States; Variant; Visual impairment; Zebrafish; base; cohort; cost effective; disability; exome; eye formation; gain of function; gene discovery; gene function; genome-wide; improved; loss of function; mortality; new technology; next generation; novel; novel strategies; proband; research study

DESCRIPTION (provided by applicant): Anophthalmia (absent eyes) is found in 1 in 5,000-10,000 live births and is closely related to microphthalmia (small eyes). It is very important that we understand the genetic factors that can predispose to anophthalmia and microphthalmia (A/M), as the health and lifestyle consequences associated with severely reduced vision can be profound. The pathogenesis of both anophthalmia and microphthalmia (A/M) is poorly understood, and more than 60% of individuals and families with A/M do not receive an underlying genetic diagnosis that explains their birth defect. Recently, next-generation sequencing, or massively parallel sequencing, has been used to discover novel genes for Mendelian syndromes and more complex disorders such as autism and intellectual disability. Next-generation sequencing has the potential to unleash significant gene discovery for birth defects, as it can be employed with a trio approach in which sequencing the exome or genomic coding regions of an affected child is performed together with exome sequencing in both unaffected, biological parents. The resulting sequence variants can be examined for novel, de novo mutations (suggestive of autosomal dominant inheritance) and genes containing more than one deleterious mutation that are inherited from separate parents (suggestive of autosomal recessive inheritance). Many genes can be rapidly screened and sequence variants can also be examined in the context of other variants in the exome. We have previously used next-generation sequencing successfully for mutation detection in known causative genes in patients with A/M and have developed a strategy for novel sequence variant identification and prioritization of candidate genes. We have also used in-situ hybridization in murine embryos to examine the expression of candidate genes in the developing eye and injections of antisense morpholinos in zebrafish to determine the ocular effects of loss of candidate gene function. In this research, we plan to extend our studies to use exome sequencing to sequence 7 trios comprising an affected child with A/M and both healthy biological parents. Two trios will be selected because the proband has A/M with an extraocular feature that is present in at least three other individuals in our patient cohort (for example, diaphragmatic hernia or choanal atresia). We will utilize public databases to prioritize novel sequence variants and verify them using Sanger sequencing. We will use our previous methodology of in-situ hybridization in mouse and morpholino or mRNA injections in Danio rerio to examine the effects of loss or gain of candidate gene function on eye development, respectively, to implicate new genes in eye formation. If we are successful in discovering new genes for A/M using next-generation technologies, our methodologies will be applicable to other birth defects and may prove to be a new paradigm for gene discovery and genetic research in non-Mendelian birth defects. PUBLIC HEALTH RELEVANCE: Birth defects affect an estimated 120,000 (1 in 33) babies born in the United States each year, and are the leading case of mortality in the first year of lif. Anophthalmia (absent eyes) and microphthalmia (small eyes) are associated with a high level of disability due to visual impairment. The genetic causes of anophthalmia and microphthalmia are poorly understood. This grant seeks to use a new sequencing technology, termed exome sequencing, to improve the existing knowledge pertaining to the genetic causes of anophthalmia and microphthalmia, so that patient counseling and management and anticipatory guidance can be improved and our understanding of eye development enhanced.

描述（由申请人提供）：无眼症（眼睛缺失）在5，000 - 10，000例活产婴儿中发现1例，与小眼症（小眼睛）密切相关。是非常重要 我们了解遗传因素可能导致无眼症和小眼症（A/M），因为与视力严重下降相关的健康和生活方式后果可能是深远的。无眼症和小眼症（A/M）的发病机制知之甚少，超过60%的A/M个体和家庭没有接受解释其出生缺陷的潜在遗传诊断。最近，下一代测序，或大规模平行测序，已被用于发现孟德尔综合征和更复杂的疾病，如自闭症和智力残疾的新基因。下一代测序有可能为出生缺陷释放重要的基因发现，因为它可以采用三重方法，其中对受影响儿童的外显子组或基因组编码区进行测序，同时对未受影响的生物学父母进行外显子组测序。可以检查所得序列变体的新的、从头突变（提示常染色体显性遗传）和含有从不同父母遗传的多于一个有害突变的基因（提示常染色体隐性遗传）。可以快速筛选许多基因，并且还可以在外显子组中的其他变体的背景下检查序列变体。我们之前已经成功地使用下一代测序技术检测A/M患者已知致病基因的突变，并开发了一种新的序列变异识别和候选基因优先级排序的策略。我们还在小鼠胚胎中使用原位杂交来检查候选基因在发育中的眼睛中的表达，并在斑马鱼中注射反义morpholinos来确定候选基因功能丧失对眼睛的影响。在这项研究中，我们计划将我们的研究扩展到使用外显子组测序来测序7个三联体，包括一个患有A/M的受影响儿童和两个健康的亲生父母。将选择两个三人组，因为先证者患有A/M，其眼外特征存在于我们的患者队列中的至少三个其他个体中（例如，疝或后鼻孔闭锁）。我们将利用公共数据库优先考虑新的序列变异，并使用桑格测序验证它们。我们将使用我们以前的方法原位杂交在小鼠和吗啉代或mRNA注射在斑马鱼检查候选基因功能的损失或获得对眼睛发育的影响，分别牵连新的基因在眼睛的形成。如果我们使用下一代技术成功发现A/M的新基因，我们的方法将适用于其他出生缺陷，并可能被证明是非孟德尔出生缺陷基因发现和遗传研究的新范式。 公共卫生相关性：据估计，出生缺陷每年影响美国出生的120，000名婴儿（33人中有1人），并且是生命第一年死亡的主要病例。无眼症（无眼）和小眼症（小眼）与视力障碍造成的高度残疾有关。无眼症和小眼症的遗传原因知之甚少。这项资助旨在使用一种新的测序技术，称为外显子组测序，以提高现有的知识有关的遗传原因的无眼和小眼，使病人的咨询和管理和预期的指导，可以改善和我们的眼睛发育的理解增强。