Harsha Rajasimha

Developing Rods Transplanted into the Degenerating Retina of Crx-Knockout Mice Exhibit Neural Activity Similar to Native Photoreceptors†‡§

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age‐related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild‐type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP‐tagged developing rods show Ca2 + responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization‐activated current (Ih) induced by the activation of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels. When transplanted into the subretinal space of wild‐type or retinal degeneration mice, GFP‐tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild‐type mouse retina and exhibit Ca2 + responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin‐positive outer segment‐like structures within 2 weeks after transplantation into the retina of Crx‐knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP‐positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod‐specific phototransduction genes, and HCN‐1 channels. We conclude that Nrl‐promoter‐driven GFP‐tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell‐derived rods in vitro may provide a renewable source for cell‐replacement therapy. STEM Cells 2013;31:1149–1159

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruchs membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruchs membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.

Retinal transcriptome profiling by directional next-generation sequencing using 100 ng of total RNA.

RNA expression profiles produced by next-generation sequencing (NGS) technology (RNA-seq) allow comprehensive investigation of transcribed sequences within a cell or tissue. RNA-seq is rapidly becoming more cost-effective for transcriptome profiling. However, its usage will expand dramatically if one starts with low amount of RNA and obtains transcript directionality during the analysis. Here, we describe a detailed protocol for the creation of a directional RNA-seq library from 100 ng of starting total RNA.

Exome sequencing: capture and sequencing of all human coding regions for disease gene discovery.

In humans, protein-coding exons constitute 1.5-1.7% of the human genome. Targeted sequencing of all coding exons is termed as exome sequencing. This method enriches for coding sequences at a genome-wide scale from 3 μg of DNA in a hybridization capture. Exome analysis provides an excellent opportunity for high-throughput identification of disease-causing variations without the prior knowledge of linkage or association. A comprehensive landscape of coding variants could also offer valuable mechanistic insights into phenotypic heterogeneity and genetic epistasis.