Danni Lin

Lanosterol reverses protein aggregation in cataracts

The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Lens regeneration using endogenous stem cells with gain of visual function

The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.

Next-generation sequencing and novel variant determination in a cohort of 92 familial exudative vitreoretinopathy patients.

PURPOSE Familial exudative vitreoretinopathy (FEVR) is a developmental disease that can cause visual impairment and retinal detachment at a young age. Four genes involved in the Wnt signaling pathway were previously linked to this disease: NDP, FDZ4, LRP5, and TSPAN12. Identification of novel disease-causing alleles allows for a deeper understanding of the disease, better molecular diagnosis, and improved treatment. METHODS Sequencing libraries from 92 FEVR patients were generated using a custom capture panel to enrich for 163 known retinal disease-causing genes in humans. Samples were processed using next generation sequencing (NGS) techniques followed by data analysis to identify and classify single nucleotide variants and small insertions and deletions. Sanger validation and segregation testing were used to verify suspected variants. RESULTS Of the cohort of 92, 45 patients were potentially solved (48.9%). Solved cases resulted from the determination of 49 unique mutations, 41 of which are novel. Of the novel variants discovered, 13 were highly likely to cause FEVR due to the nature of these variants (frameshifting indels, splicing mutations, and nonsense variants types). To our knowledge, this is the largest study of a FEVR cohort using NGS. CONCLUSIONS We were able to determine probable disease-causing variants in a large number of FEVR patients, the majority of which were novel. Knowledge of these variants will help to further characterize and diagnose FEVR.

DNA methylation markers for diagnosis and prognosis of common cancers

Significance The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving diagnosis and prognosis. We evaluated the utility of DNA methylation profiles for differentiating tumors and normal tissues for four common cancers (lung, breast, colon, and liver) and found that they could differentiate cancerous tissue from normal tissue with >95% accuracy. This signature also correctly identified 19 of 20 breast cancer metastases and 29 of 30 colorectal cancer metastases to the liver. We report that methylation patterns can predict the prognosis and survival, with good correlation between differential methylation of CpG sites and expression of cancer-associated genes. Their findings demonstrate the utility of methylation biomarkers for the molecular characterization, diagnosis, and prognosis of cancer. The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving the diagnosis, treatment selection, and prediction of prognosis in cancer. Using whole-genome methylation data from The Cancer Genome Atlas (TCGA) and machine learning methods, we evaluated the utility of DNA methylation for differentiating tumor tissue and normal tissue for four common cancers (breast, colon, liver, and lung). We identified cancer markers in a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples. We replicated our findings in a separate TCGA cohort of 791 tumor samples and 93 matched adjacent normal tissue samples, as well as an independent Chinese cohort of 394 tumor samples and 324 matched adjacent normal tissue samples. The DNA methylation analysis could predict cancer versus normal tissue with more than 95% accuracy in these three cohorts, demonstrating accuracy comparable to typical diagnostic methods. This analysis also correctly identified 29 of 30 colorectal cancer metastases to the liver and 32 of 34 colorectal cancer metastases to the lung. We also found that methylation patterns can predict prognosis and survival. We correlated differential methylation of CpG sites predictive of cancer with expression of associated genes known to be important in cancer biology, showing decreased expression with increased methylation, as expected. We verified gene expression profiles in a mouse model of hepatocellular carcinoma. Taken together, these findings demonstrate the utility of methylation biomarkers for the molecular characterization of cancer, with implications for diagnosis and prognosis.

A missense mutation in HK1 leads to autosomal dominant retinitis pigmentosa.

PURPOSE Retinitis pigmentosa (RP) is a genetically heterogeneous disease with over 60 causative genes known to date. Nevertheless, approximately 40% of RP cases remain genetically unsolved, suggesting that many novel disease-causing genes are yet to be identified. In this study, we aimed to identify the causative mutation for a large autosomal dominant RP (adRP) family with negative results from known retinal disease gene screening. METHODS Linkage analysis followed by whole-exome sequencing was performed. Stringent variant filtering and prioritization was carried out to identify the causative mutation. RESULTS Linkage analysis identified a minimal disease region of 8 Mb on chromosome 10 with a peak parametric logarithm (base 10) of odds (LOD) score of 3.500. Further whole-exome sequencing identified a heterozygous missense mutation (NM_000188.2:c.2539G>A, p.E847K) in hexokinase 1 (HK1) that segregated with the disease phenotype in the family. Biochemical assays showed that the E847K mutation does not affect hexokinase enzymatic activity or the protein stability, suggesting that the mutation may impact other uncharacterized function or result in a gain of function of HK1. CONCLUSIONS Here, we identified HK1 as a novel causative gene for adRP. This is the first report that associates the glucose metabolic pathway with human retinal degenerative disease, suggesting a potential new disease mechanism.

Transcription Factor PAX6 (Paired Box 6) Controls Limbal Stem Cell Lineage in Development and Disease

Background: PAX6 is a master regulatory gene involved in eye development. Results: PAX6 is expressed in the primitive eye cup and later in corneal tissue progenitors in early embryonic development. Conclusion: PAX6 plays a critical role in limbal stem cell and corneal epithelial fate determination. Significance: These findings provide important insight into corneal homeostasis and disease. PAX6 is a master regulatory gene involved in neuronal cell fate specification. It also plays a critical role in early eye field and subsequent limbal stem cell (LSC) determination during eye development. Defects in Pax6 cause aniridia and LSC deficiency in humans and the Sey (Small eye) phenotype in mice (Massé, K., Bhamra, S., Eason, R., Dale, N., and Jones, E. A. (2007) Nature 449, 1058–1062). However, how PAX6 specifies LSC and corneal fates during eye development is not well understood. Here, we show that PAX6 is expressed in the primitive eye cup and later in corneal tissue progenitors in early embryonic development. In contrast, p63 expression commences after that of PAX6 in ocular adnexal and skin tissue progenitors and later in LSCs. Using an in vitro feeder-free culture system, we show that PAX6 knockdown in LSCs led to up-regulation of skin epidermis-specific keratins concomitant with differentiation to a skin fate. Using gene expression analysis, we identified the involvement of Notch, Wnt, and TGF-β signaling pathways in LSC fate determination. Thus, loss of PAX6 converts LSCs to epidermal stem cells, as demonstrated by a switch in the keratin gene expression profile and by the appearance of congenital dermoid tissue.

Corrigendum: Lanosterol reverses protein aggregation in cataracts

This corrects the article DOI: 10.1038/nature14650

Corrigendum: Lens regeneration using endogenous stem cells with gain of visual function

This corrects the article DOI: 10.1038/nature17181

Examination of the Retina

Indications The retinal examination is part of a complete physical examination. It is particularly important in patients with a history of such systemic diseases as diabetes, hypertension, and atherosclerosis. Patients with the human immunodeficiency virus (HIV) or the acquired immunodeficiency syndrome (AIDS) are also at increased risk for retinal diseases. Evaluation of the retina may reveal the initial signs of diabetic retinopathy, hypertensive retinopathy, macular edema, glaucoma, or macular degeneration. Diagnosis and appropriate interventions can help preserve sight and prevent disease progression. 2 Indications for urgent direct ophthalmoscopy include a clinical suspicion of increased intracranial pressure, occluded retinal vessels, and retinal detachment. Any of these conditions, or the sudden loss of vision or a change in vision, constitutes an ophthalmic emergency. The patient should be seen by an ophthalmologist immediately or should be sent to the emergency department. Anatomy The inner structures of the eye can be visualized through the pupil, the central opening in the iris. The light rays from the ophthalmoscope pass through the cornea, pupil, and lens to focus on the retina, producing an upright, magnified image. The retina is located on the inner surface of the globe, opposite the pupillary opening. The color should appear red, orange, or brown and varies from person to person. Direct Ophthalmoscopic Settings The direct ophthalmoscope consists of a handle, which contains the power supply and a light source, and a head, which contains the viewing window and lenses. The on–off switch adjusts the brightness of the light. The apertures include a large circle, a medium circle, a small circle, and a slit beam. To reduce the patient’s pupillary constriction it is best to use the ophthalmoscope at a brightness level of 80 to 90%, with the aperture set to the small or medium circle. The ophthalmoscope contains a range of lenses with positive and negative diopters that compensate for refractive error on the part of the examiner and the patient. If the patient has hyperopia, the retina will be closer to the pupil than normal. If the patient has myopia, the retina will be farther away from the pupil