Paul A. Sieving, MD PhD - US grants

This project seeks to study electroretinogram (ERG) components that derive from the inner retina. The ERG is an extracellular response of the retina to stimulation by light and has traditionally been considered to reflect primarily outer retinal origins. As such it is currently used clinically in diagnosing human eye disease of the photoreceptors and structures nearby, such as in retinitis pigmentosa. However, more recently it has been appreciated that ERG components from the inner retina can also be recorded with appropriate stimuli, both within the retina using microelectrodes and clinically at the cornea. As a basic research tool, these responses may yield insight into how the inner retina functions. These proximal responses also greatly extend the scope of ERG testing and may provide an important new means to monitor human inner retinal pathology in glaucoma, diabetic and hypertensive retinopathy, and possibly amblyopia. Three ERG responses that have proximal retinal components will be investigated: the pattern-ERG, the photopic On- and Off-components, and the scotopic negative response below b-wave threshold. The aim is to characterize these responses in detail and to study the precise retinal depths of origin. The research strategy involves first studying the ERG in a convenient animal model, the cat. Intraretinal recording will be performed to identify the extra-cellular current source/sinks so that ultimately they can be linked to cell types. A second approach will evaluate how these ERG responses are changed in a cat model of inner retinal pathology; this will help clarify the origins of these components and may provide clues applicable in diagnosing human disease. Third, a correlated study of human ERG recordings will be performed to understand the normal response range; then, recordings will be evaluated in human ertinal disease to distinguish inner from outer retinal ERG sources in man and to begin clarifying the clinical value of these tests. The long term goal is to learn which retinal cells generate these responses and by what mechanism. This knowledge will increase our fundamental understanding of information processing in the retina, especially from a region that is otherwise difficult to monitor except by intracellular recordings. This knowledge will also increase the power of clinical ERG tests to help establish the locus of some human retinal diseases and to provide clues about their nature.

Our goal is to understand further the genetic and development basis of human X-linked juvenile retinoschisis (RS) (#31270 in McKusick, 1992) and the biology of Muller cells (retinal glial cells) that are believed to be involved in RS. This proposal presents the positional cloning strategy that we are using to identify and clone the RS gene. Retinoschisis causes significant loss of vision from a young age from macular cysts that are present at birth. In the peripheral retina the inner layers peep from the outer layers peel from the outer layers like sheets of tissue paper and can lead to unrepairable retinal detachment. The carrier state is not detectable, and cloning the RS gene will immediately benefit genetic counseling. The biological importance of RS extends beyond the clinical implications and involves elucidating mechanisms of retinal pathology presumed to be caused by Muller cell dysfunction. RS presents a unique opportunity to study inner retinal pathophysiology, since, unlike most other human inherited retinal dystrophies that affect the outer retinal layers, RS affects the proximal retina. In later age, RS males typically show secondary atrophy of the retinal pigment epithelium, and RS may provide insight into mechanisms important for dry forms of Age-Related Macular Degeneration. Muller cells impart structural integrity to the retina and mediate the retinal extracellular ionic homeostasis that is critical for function and survival of retinal neurons. Understanding the genetic and biological basis of RS holds promise of new understanding of basic mechanisms important for vision and retinal function. Our preliminary RS genetic study involved linkage with seven large families for which flanking RFLP markers were identified in p22.1-p22.3 (Sieving et al, 1990). We have now collected DNA from 35 RS pedigrees that include 102 affected males and more than 250 scorable meioses. We are cloning a portion of Xp22.1-22.3 into YAC contigs, preparatory to elaborating a micro-scale genetic and physical map around the RS locus. This project participants in the Michigan Human Genome Initiative Center which provides access to specialized technical support and resources. We have screened YAC libraries at the Michigan Genome Center using the RFLP marker closest to RS and have obtained four different YAC clones which we are now characterizing to construct a YAC contig around RS. We will then screen the contig for new microsatellite markers for genetic testing against RS. We are laos using a candidate gene approach by testing cDNAs in the RS region from a human retinal library enriched for X-chromosomal cDNAs.

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this research is to understand the developmental and genetic basis for human X-linked juvenile RS. RS causes significant vision loss due to macular degeneration beginning in infancy and progressive changes in later age mimic age-related macular degeneration. Peripheral vision is also affected due to splitting of the retina through the nerve fiber layer which lies at the surface of the retina. Muller cells (retinal glial cells) are believed to be affected in RS, and this project will help elucidate some of the biology of these retinal glial cells. This proposal is to identify the molecular defect for RS by a positional cloning approach. The research plan encompasses five specific aims. Aim one is to continue collecting RS families, to narrow the RS inclusion-interval. These will be used to find additional crossovers and potentially new deletions in the RS region. Aim two is to clone the RS gene, which appears to be within a 950 kb critical region, contained on a single 2.2 Mb YAC clone (939h7). A cosmid sublibrary has been made from this clone, and the cosmids are being assembled into a contig. Transcribed segments are being isolated by exon-trapping, and selection-screening of cDNA. Candidate genes will be tested by amplifying exons from multiple affected and unaffected controls, and searching for deletions and/or point mutations by sequencing. The applicant is also evaluating connexin-33, which maps to the critical region, as a candidate gene. Aim three is to determine the pattern of gene expression in retina, once the RS gene has been cloned. This will involve in situ hybridization and immunocytochemistry, using antibody against fusion proteins. Aim four is to correlate the genotype with the phenotype in the different RS alleles. Phenotypic features to correlate include severity at birth, progression, and presence of macular degeneration features. Aim five is to test the relationship between the ERG b-wave generator and the RS gene product.