Molecular Genetics of Inherited Retinal DisordersInherited retinal degenerations such as retinitis pigmentosa (RP) are common causes of blindness. The overall goals of our research program are to improve our understanding of the molecular bases of inherited retinal degenerations and related cilia disorders so that rational therapies can be developed for these disease.
As described below, we currently have 7 active research projects directed towards these goals.
An important focus of the lab’s work is the discovery of new genetic defects leading to different forms of inherited retinal degenerations (IRDs). These include non-syndromic diseases such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Cone (CD) and Cone-rod dystrophies (CRD) and syndromic forms such as Usher Syndrome and ciliopathies (e.g. Joubert, Senior-Loken, Bardet-Biedl syndromes). These diseases are very heterogeneous and so far have been associated with mutations in approximately 200 genes (the full list of genes can be found at https://sph.uth.edu/retnet/disease.htm#17.105d). However, it has been estimated that mutations in known IRD genes account for only 50-60% of cases and therefore much effort is still needed to discover the remaining genetic defects.
To identify the new IRD associated genes we integrate the techniques of whole-exome sequencing, copy number variation (CNV) analysis, linkage and homozygosity mapping using SNP arrays. For these analyses we select patients that have been previously excluded for mutations in known IRD genes by a targeted next generation sequencing and CGH array studies. We conduct two strategies of novel gene discovery: 1) family genetics and 2) cohort based study. In family genetics we analyze all available family members and look for rare, likely pathogenic genetic variants that segregate with the phenotype. Examples of such families are presented in Figure 1. In the cohort based studies we select patients of similar phenotype, for which we do not have available family members, and analyze them together as a group. Here we search for common rare, likely pathogenic variants between the affected individuals. At the moment we study three distinct cohorts: Usher type I, LCA and pericentral RP patients.
Figure 1. Example pedigrees of families selected for whole-exome sequencing without mutations in known IRD disease genes. Probands (P) are indicated. JS, Joubert syndrome.
Part of our work on PSCs is focused on the retinitis pigmentosa 1 (RP1) protein. Mutations in RP1 are a common cause of dominant RP, which is the most common form of inherited retinal degeneration. Work in our lab has found that the RP1 protein is a photoreceptor microtubule-associated protein that is required for the correct formation of PSCs. We are now working to identify proteins that interact with RP1 in order to further define how it participates in PSC formation, and study how its mutations lead to photoreceptor cell death. We are also beginning to test potential therapies for RP1 disease, including gene augmentation therapy, in point mutation Rp1 knock-in mice.
We and others recently reported that mutations in the NMNAT1 gene are a common cause of Leber congenital amaurosis (LCA), accounting for ~5% of cases (1-5). NMNAT1 encodes an essential enzyme that generates NAD+ both in a biosynthetic pathway from nicotinic acid mononucleotide (NaMN) and in a salvage pathway from nicotinamide mononucleotide (NMN) (6). Three functionally non-redundant mammalian NMNAT isoforms encoded by different genes have been identified within distinct cellular compartments, where NMNAT1, 2 and 3 localize, respectively, to the nucleus, Golgi complex, and mitochondria (7,8). The mitochondrial isoform, NMNAT3, regenerates NAD+ for cellular energetics, whereas NMNAT1 is involved in nuclear NAD+ homeostasis necessary for both DNA metabolism and cell signaling (Figure 1) (8).
The identification of NMNAT1 as an LCA disease gene raises the intriguing question of how mutations in a widely expressed NAD+ biosynthetic protein lead to a retina-specific phenotype. The answer to this question is of particular interest because the majority of patients with NMNAT1-LCA have atrophic macular lesions, suggesting that macular cones are especially dependent upon NMNAT1 activity (Figure 2) (2-5). Data from studies performed to date suggest the mutations identified in NMNAT1 lead to decreased NAD+ biosynthetic activity (2,3). We therefore hypothesize that the retinal degeneration caused by mutations in NMNAT1 results primarily from decreased nuclear NAD+ synthesis and the associated alteration in NAD+ homeostasis in the nuclei of retinal cells. This hypothesis also suggests that therapies directed at restoring NAD+ biosynthesis should be beneficial for NMNAT1 LCA. Since cells with decreased NMNAT1 enzyme activity do not appear to be able to utilize NAD+ precursors to increase NAD+ levels, increasing NMNAT1 enzyme activity via gene therapy is an attractive treatment approach (2).
Of interest, NMNAT1 is the principle component of the Wallerian degeneration slow (WldS) fusion protein, which also includes a 70 amino acid N-terminal sequence from the Ube4b multi-ubiquitination factor. The chimeric WldS gene and protein were identified through study of a line of spontaneous mutant mice in which axon stumps that are distal to an injury survive ten times longer than normal; that is, the mice exhibit slow Wallerian degeneration (9-11). It has since been determined that the axonal protection activity of the WldS protein in mice requires both the Ube4b component and an enzymatically active Nmnat1 portion of the chimeric protein (12). Further, expression of the normally nuclear Nmnat1 protein in extranuclear locations can protect axons, suggesting that extranuclear Nmnat enzymatic activity is required for axon protection (13-16). Despite the robust effects of the WldS protein on axon degeneration, it does not appear that WldS protects neuron cell bodies from degeneration (9). For example, retinal ganglion cell axons in WldS mice are protected following induction of glaucoma or optic nerve crush, but RGC cell bodies are not (17,18). Since the retinal degeneration caused by mutations in NMNAT1 causes notable retinal cell loss, especially in the macula, we are focusing our experiments on the potential role of nuclear NAD+ in retinal biology, rather than on the axon protection activity of NMNAT1.
We are currently:
- Investigating the pathogenesis of NMNAT1 disease via biochemical studies and gene targeted Nmnat1 mutant mice
- Working to develop adeno-associated virus (AAV)-mediated gene augmentation therapy for NMNAT1 disease
Nature Genetics publication: http://www.ncbi.nlm.nih.gov/pubmed/22842227
1. den Hollander AI, Black A, Bennett J, Cremers FP. Lighting a candle in the dark: advances in genetics and gene therapy of recessive retinal dystrophies. The Journal of clinical investigation. 2010;120(9):3042-53. PMCID: 2929718.
Cilia are present on most cells in the human body. These structures are typically sensory organelles, and are involved in many critical aspects of cell biology and development. The photoreceptor sensory cilium (PSC) elaborated by each rod and cone photoreceptor cell of the retina is a classic example (Figure 1). Consistent with the importance of cilia in biology, mutations in genes that encode cilia components are common causes of disease. Mutations that cause inherited retinal degenerations, which are common causes of blindness, have been identified in genes encoding more than 40 PSC proteins to date. These disorders are characterized by PSC dysfunction, followed by degeneration and death of the photoreceptor cells, resulting in loss of vision.
We are interested in studying how photoreceptor sensory cilia are built and maintained, and how these processes are disrupted in disease. For example, while there has been notable progress identifying the genetic causes of inherited retinal degenerations and other cilia disorders, the genes that harbor mutations which cause disease in half of patients with inherited retinal degeneration remain to be identified. To help understand PSCs better, and facilitate identification of new retinal degeneration disease genes, we performed a series of proteomic analyses to identify all of the proteins in mouse photoreceptor sensory cilia. The results show that PSCs are made of almost 2000 proteins, including ~1500 proteins not detected in cilia from lower organisms. This database of PSC proteins has already proved to be very useful. For example, in the past year we have used the list of genes that encode novel PSC proteins to help identify one confirmed and four potential new retinal degeneration disease genes.
Mature RNA transcripts are produced by the complex, and dynamic, process of removing introns from a nascent transcript while splicing the remaining exons together. RNA splicing is mediated by the spliceosome, a multi-component macromolecule, inside the nucleus during transcription (REFs). The number of genes encoded in any genome is minimal, relative to the number of proteins produced, which is due to alternative splicing (Refs). In humans, 94-96% of transcripts are alternatively spliced producing multiple isoforms of each transcript; thus, providing the complexity observed in the proteome.
RNA splicing is essential, and occurs, in every eukaryotic cell (Refs). It is of particular interest to our group to study why mutations in proteins found in the spliceosome cause non-syndromic retinitis pigmentosa (RP) (Refs). Currently, mutations in PRPF3, PRPF6, PRPF8, PRPF31, SNRNP200, and RP9 have been found to cause RP (Refs). All of the proteins are located in the U4/U6-U5 tri-snRNP of the spliceosome (Figure 1). Given the importance of splicing in every cell type of the human body, we ask why mutations in these splicing factors only affect vision?
To investigate this question, we have developed knock-in mouse models mimicking the human mutations found in PRPF3 and PRPF8, as well as a knock-out mouse model of PRPF31. Phenotypic characterization of these models suggests the RPE is the primary site of pathogenesis for each of these models. The RPE undergoes morphological changes with a loss of basal infoldings and vacuolization at 2 years of age. Phagocytosis assays of primary RPE cultures suggest a loss of functional activity in 2 week-old mice. This phagocytosis deficiency has also been observed in shRNA-mediated knockdown of PRPF31 in aRPE-19 cell culture.
If the RPE is the primary site of pathogenesis, what is the cause of this cellular specificity? Since components of the spliceosome are affected, we hypothesize that aberrant splicing is the cause of pathogenesis in the RPE. We have employed RNA-Seq to study the transcriptomes of the RPE, neural retina, brain, and skeletal muscle in the mouse models.
We are currently:
- Performing a detailed analysis of the RPE phenotype by investigating the functional changes in the mutant mouse models.
- Analyzing the transcriptomes of the mouse models to characterize the effects of mutations in the Prpf splicing factors on splicing and disease pathogenesis.
Age-related macular degeneration (AMD) is one of the most common cause of vision loss in developed countries. The most characteristic clinical finding in the retinas of patients with AMD is drusen, or extracellular deposits of protein, lipid and debris that accumulate underneath the retinal pigment epithelium (RPE). At present, the etiology of drusen in AMD is not known, and there are only limited treatments are available to prevent the progression of AMD. In order to gain insight into the pathogenesis of AMD, we are studying an inherited form of macular degeneration called Doyne honeycomb retinal dystrophy (DHRD)/Malattia Leventinese (ML). Both DHRD and ML are caused by a single mutation, Arg-345 to Trp (R345W), in the EFEMP1 or Fibulin-3 gene. We have used gene targeting techniques to introduce this mutation into the Efemp1 gene of mice. We have found that the Efemp1-R345W knock-in mice develop AMD-like deposits under their retinas, and are now using proteomic analyses to study the pathogenesis of these lesions.