AUTOIMMUNE RETINOPATHY MIMICKING HERITABLE RETINAL DEGENERATION IN A PATIENT WITH COMMON VARIABLE IMMUNE DEFICIENCY.

PURPOSE: 1) To describe a case of autoimmune retinopathy mimicking heritable photoreceptor degeneration in a patient with common variable immune deficiency and 2) to investigate the humoral and cell-mediated branches of the immune system in this patient to better understand the mechanism of immune-mediated photoreceptor damage in this disease. METHODS: Retrospective chart review with evaluation of multimodal imaging, genotype analysis, and investigation of circulating autoantibodies and T-cell response to retinal antigens. RESULTS: A 40-year-old woman with bilateral, progressive vision loss was referred for evaluation of a possible inherited retinal degeneration. She was found to have asymmetric peripheral visual field constriction, cystoid macular edema, vitreous cells, and bone spicule-like pigmentary changes in both eyes. An extensive workup for underlying infectious or inflammatory causes was unrevealing, and molecular analysis for heritable retinal degeneration failed to identify a plausible disease-causing genotype. Screening for antiretinal antibodies showed the presence of multiple antiretinal antibodies, consistent with a diagnosis of autoimmune retinopathy. Immunologic workup demonstrated markedly decreased levels of serum IgA and IgG, consistent with common variable immune deficiency. T-cells isolated from the patient showed increased proliferation when stimulated with human retinal proteins, supporting a role for both cell- and humoral-mediated autoimmunity. Treatment with mycophenolate mofetil and intravenous immunoglobin therapy slowed the progression of disease and resulted in preservation of her central vision. CONCLUSION: Autoimmune retinopathy can be seen in common variable immune deficiency and has clinical findings similar to heritable photoreceptor degeneration. Both the humoral and cellular immune responses are involved in the pathophysiology. Immune modulatory therapy has stabilized the disease course in this patient and may play an important role in the management of autoimmune retinopathy.

Label-free microfluidic enrichment of cancer cells from non-cancer cells in ascites.

The isolation of a patient's metastatic cancer cells is the first, enabling step toward treatment of that patient using modern personalized medicine techniques. Whereas traditional standard-of-care approaches select treatments for cancer patients based on the histological classification of cancerous tissue at the time of diagnosis, personalized medicine techniques leverage molecular and functional analysis of a patient's own cancer cells to select treatments with the highest likelihood of being effective. Unfortunately, the pure populations of cancer cells required for these analyses can be difficult to acquire, given that metastatic cancer cells typically reside in fluid containing many different cell populations. Detection and analyses of cancer cells therefore require separation from these contaminating cells. Conventional cell sorting approaches such as Fluorescence Activated Cell Sorting or Magnetic Activated Cell Sorting rely on the presence of distinct surface markers on cells of interest which may not be known nor exist for cancer applications. In this work, we present a microfluidic platform capable of label-free enrichment of tumor cells from the ascites fluid of ovarian cancer patients. This approach sorts cells based on differences in biomechanical properties, and therefore does not require any labeling or other pre-sort interference with the cells. The method is also useful in the cases when specific surface markers do not exist for cells of interest. In model ovarian cancer cell lines, the method was used to separate invasive subtypes from less invasive subtypes with an enrichment of ~ sixfold. In ascites specimens from ovarian cancer patients, we found the enrichment protocol resulted in an improved purity of P53 mutant cells indicative of the presence of ovarian cancer cells. We believe that this technology could enable the application of personalized medicine based on analysis of liquid biopsy patient specimens, such as ascites from ovarian cancer patients, for quick evaluation of metastatic disease progression and determination of patient-specific treatment.

Wide-Field Swept-Source OCT and Angiography in X-Linked Retinoschisis.

PURPOSE: Retinal vascular and structural changes, particularly outside of the central macula, are not well characterized in X-linked retinoschisis (XLRS). We aim to describe wide-field swept-source OCT (SS-OCT) and swept-source OCT angiography (SS-OCTA) findings in XLRS. DESIGN: Retrospective, cross-sectional study at a tertiary referral center. PARTICIPANTS: Nine consecutive male patients with molecularly confirmed XLRS. METHODS: All patients underwent complete ophthalmic examination with multimodal imaging, including SS-OCT with SS-OCTA (PLEX Elite 9000; Carl-Zeiss Meditec Inc., Dublin, CA). Images were then reviewed by 2 retinal specialists as independent graders to determine the frequency and distribution of retinal structural and vascular abnormalities. MAIN OUTCOME MEASURES: Structural and vascular abnormalities seen on SS-OCT and SS-OCTA in patients with XLRS, with attention to the retinal layers involved, the regional distribution of schitic spaces in the posterior pole, and vascular abnormalities within the superficial and deep capillary plexuses. RESULTS: Eighteen eyes from 9 male patients (mean age, 20 years; range 9-40) with molecularly confirmed XLRS were included. Median best-corrected visual acuity measured 20/63 (range, 20/25-10/300). A total of 17 of 18 eyes (94.4%) were noted to have schitic spaces on SS-OCT, and these were observed to be predominantly within the inner nuclear layer in all 17 eyes. A regional variation in the distribution of cysts was noted, with schitic spaces within the ganglion cell layer (13/17 eyes; 76.5%) observed to be perifoveal and those within the outer nuclear layer (8/17 eyes, 47.1%) observed to be mostly extramacular. All eyes had vascular abnormalities on SS-OCTA, including an irregular foveal avascular zone and flow loss within the deep capillary plexus corresponding to the distribution of the schisis. CONCLUSIONS: Wide-field SS-OCT and SS-OCTA provide detailed visualization of structural and vascular changes in XLRS and may be helpful for monitoring disease progression or treatment response in clinical trials for the disease.

Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease.

PURPOSE: To devise a comprehensive multiplatform genetic testing strategy for inherited retinal disease and to describe its performance in 1000 consecutive families seen by a single clinician. DESIGN: Retrospective series. PARTICIPANTS: One thousand consecutive families seen by a single clinician. METHODS: The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed, and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to 1 of 62 diagnostic categories, and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from 2 to nearly 900 000. MAIN OUTCOME MEASURES: Sensitivity and false genotype rate. RESULTS: Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 75% of these 104 genes have coding sequences small enough to be packaged efficiently into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families), whereas mutations in 80 genes caused disease in 5 or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next-generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole-exome sequencing of the remaining 424 families revealed mutations in an additional 182 families, and whole-genome sequencing of 4 of the remaining 242 families revealed 2 additional genotypes that were invisible by the other methods. Performing the testing in a clinically focused tiered fashion would be 6.1% more sensitive and 17.7% less expensive and would have a significantly lower average false genotype rate than using whole-exome sequencing to assess more than 300 genes in all patients (7.1% vs. 128%; P < 0.001). CONCLUSIONS: Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.

Primary congenital and developmental glaucomas.

Glaucoma is the leading cause of irreversible blindness worldwide. Although most glaucoma patients are elderly, congenital glaucoma and glaucomas of childhood are also important causes of visual disability. Primary congenital glaucoma (PCG) is isolated, non-syndromic glaucoma that occurs in the first three years of life and is a major cause of childhood blindness. Other early-onset glaucomas may arise secondary to developmental abnormalities, such as glaucomas that occur with aniridia or as part of Axenfeld-Rieger syndrome. Congenital and childhood glaucomas have strong genetic bases and disease-causing mutations have been discovered in several genes. Mutations in three genes (CYP1B1, LTBP2, TEK) have been reported in PCG patients. Axenfeld-Rieger syndrome is caused by mutations in PITX2 or FOXC1 and aniridia is caused by PAX6 mutations. This review discusses the roles of these genes in primary congenital glaucoma and glaucomas of childhood.

cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

Prediction of cochlear implant performance by genetic mutation: the spiral ganglion hypothesis.

BACKGROUND: Up to 7% of patients with severe-to-profound deafness do not benefit from cochlear implantation. Given the high surgical implantation and clinical management cost of cochlear implantation (>$1 million lifetime cost), prospective identification of the worst performers would reduce unnecessary procedures and healthcare costs. Because cochlear implants bypass the membranous labyrinth but rely on the spiral ganglion for functionality, we hypothesize that cochlear implant (CI) performance is dictated in part by the anatomic location of the cochlear pathology that underlies the hearing loss. As a corollary, we hypothesize that because genetic testing can identify sites of cochlear pathology, it may be useful in predicting CI performance. METHODS: 29 adult CI recipients with idiopathic adult-onset severe-to-profound hearing loss were studied. DNA samples were subjected to solution-based sequence capture and massively parallel sequencing using the OtoSCOPE(®) platform. The cohort was divided into three CI performance groups (good, intermediate, poor) and genetic causes of deafness were correlated with audiometric data to determine whether there was a gene-specific impact on CI performance. RESULTS: The genetic cause of deafness was determined in 3/29 (10%) individuals. The two poor performers segregated mutations in TMPRSS3, a gene expressed in the spiral ganglion, while the good performer segregated mutations in LOXHD1, a gene expressed in the membranous labyrinth. Comprehensive literature review identified other good performers with mutations in membranous labyrinth-expressed genes; poor performance was associated with spiral ganglion-expressed genes. CONCLUSIONS: Our data support the underlying hypothesis that mutations in genes preferentially expressed in the spiral ganglion portend poor CI performance while mutations in genes expressed in the membranous labyrinth portend good CI performance. Although the low mutation rate in known deafness genes in this cohort likely relates to the ascertainment characteristics (postlingual hearing loss in adult CI recipients), these data suggest that genetic testing should be implemented as part of the CI evaluation to test this association prospectively.

Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with alpha-tectorin and is mutated in autosomal dominant hearing loss (DFNA4).

We report on a secreted protein found in mammalian cochlear outer hair cells (OHC) that is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of adhesion proteins. Ceacam16 mRNA is expressed in OHC, and its protein product localizes to the tips of the tallest stereocilia and the tectorial membrane (TM). This specific localization suggests a role in maintaining the integrity of the TM as well as in the connection between the OHC stereocilia and TM, a linkage essential for mechanical amplification. In agreement with this role, CEACAM16 colocalizes and coimmunoprecipitates with the TM protein α-tectorin. In addition, we show that mutation of CEACAM16 leads to autosomal dominant nonsyndromic deafness (ADNSHL) at the autosomal dominant hearing loss (DFNA4) locus. In aggregate, these data identify CEACAM16 as an α-tectorin-interacting protein that concentrates at the point of attachment of the TM to the stereocilia and, when mutated, results in ADNSHL at the DFNA4 locus.

Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing.

The extreme genetic heterogeneity of nonsyndromic hearing loss (NSHL) makes genetic diagnosis expensive and time consuming using available methods. To assess the feasibility of target-enrichment and massively parallel sequencing technologies to interrogate all exons of all genes implicated in NSHL, we tested nine patients diagnosed with hearing loss. Solid-phase (NimbleGen) or solution-based (SureSelect) sequence capture, followed by 454 or Illumina sequencing, respectively, were compared. Sequencing reads were mapped using GSMAPPER, BFAST, and BOWTIE, and pathogenic variants were identified using a custom-variant calling and annotation pipeline (ASAP) that incorporates publicly available in silico pathogenicity prediction tools (SIFT, BLOSUM, Polyphen2, and Align-GVGD). Samples included one negative control, three positive controls (one biological replicate), and six unknowns (10 samples total), in which we genotyped 605 single nucleotide polymorphisms (SNPs) by Sanger sequencing to measure sensitivity and specificity for SureSelect-Illumina and NimbleGen-454 methods at saturating sequence coverage. Causative mutations were identified in the positive controls but not in the negative control. In five of six idiopathic hearing loss patients we identified the pathogenic mutation. Massively parallel sequencing technologies provide sensitivity, specificity, and reproducibility at levels sufficient to perform genetic diagnosis of hearing loss.