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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Genetics of Health and Disease Study Section (GHD)
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Araj, Houmam H
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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Lamont, Ryan E; Tan, Wen-Hann; Innes, A Micheil et al. (2016) Expansion of phenotype and genotypic data in CRB2-related syndrome. Eur J Hum Genet 24:1436-44
Wu, Di; Mandal, Shyamali; Choi, Alex et al. (2015) DLX4 is associated with orofacial clefting and abnormal jaw development. Hum Mol Genet 24:4340-52
Slavotinek, A M; Garcia, S T; Chandratillake, G et al. (2015) Exome sequencing in 32 patients with anophthalmia/microphthalmia and developmental eye defects. Clin Genet 88:468-73
Choi, Alex; Lao, Richard; Ling-Fung Tang, Paul et al. (2015) Novel mutations in PXDN cause microphthalmia and anterior segment dysgenesis. Eur J Hum Genet 23:337-41