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.
Retrospective ...series.
One thousand consecutive families seen by a single clinician.
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.
Sensitivity and false genotype rate.
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).
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.
To identify specific mutations causing North Carolina macular dystrophy (NCMD).
Whole-genome sequencing coupled with reverse transcription polymerase chain reaction (RT-PCR) analysis of gene ...expression in human retinal cells.
A total of 141 members of 12 families with NCMD and 261 unrelated control individuals.
Genome sequencing was performed on 8 affected individuals from 3 families affected with chromosome 6–linked NCMD (MCDR1) and 2 individuals affected with chromosome 5–linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies <1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in 8 additional MCDR1 families and 261 controls. The RT-PCR analysis of selected genes was performed in stem cell–derived human retinal cells.
Co-segregation of rare genetic variants with disease phenotype.
Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants spanning 1 Mb of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in 5 additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1 kb centered on V1 was performed in the remaining 4 NCMD probands, and 2 additional novel single nucleotide variants (V2 in 3 families and V3 in 1 family) were identified in the DHS within 134 bp of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). The RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next-generation sequencing of 2 individuals with MCDR3-linked NCMD revealed a 900-kb duplication that included the entire IRX1 gene (V5). The 5 mutations V1 to V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families.
We identified 5 rare mutations, each capable of arresting human macular development. Four of these strongly implicate the involvement of PRDM13 in macular development, whereas the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.
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.
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 (Nimble-Gen) 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.
Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in ...exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK–associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.
Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of ...Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10(-6)). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing.
Single-cell RNA sequencing has revolutionized ocular gene expression studies. This technology has enabled researchers to identify expression signatures for rare cell types and characterize how gene ...expression changes across biological conditions, such as topographic region or disease status. However, sharing single-cell RNA sequencing results remains a major obstacle, particular for individuals without a computational background. To address these limitations, we developed Spectacle, an interactive web-based resource for exploring previously published single-cell RNA sequencing data from ocular studies. Spectacle is powered by a locally developed R package, cellcuratoR, which utilizes the Shiny framework in R to generate interactive visualizations for single-cell expression data. Spectacle contains five pre-processed ocular single-cell RNA sequencing data sets and is accessible via the web at OcularGeneExpression.org/singlecell. With Spectacle, users can interactively identify which cell types express a gene of interest, detect transcriptomic subpopulations within a cell type, and perform highly flexible differential expression analyses. The freely-available Spectacle system reduces the bioinformatic barrier for interacting with rich single-cell RNA sequencing studies from ocular tissues, making it easy to quickly identify cell types that express a gene of interest.
•Spectacle is a resource for exploring ocular single-cell RNA sequencing datasets.•Spectacle includes five datasets from the retina, RPE, and choroid.•Spectacle facilitates interactive expression analyses on pre-classified cell types.•Spectacle is freely available at OcularGeneExpression.org/singlecell.
Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis ...pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes.
•We examined gene expression in temporal, macular, and nasal regions of human retina and RPE/choroid using RNA-Seq.•Expression differences between macula and periphery in both tissues reflect the distribution of cell types.•Nasal and temporal regions of neural retina are indistinguishable in our analysis.
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.
The human neural retina is a light sensitive tissue with remarkable spatial and cellular organization. Compared with the periphery, the central retina contains more densely packed cone photoreceptor ...cells with unique morphologies and synaptic wiring. Some regions of the central retina exhibit selective degeneration or preservation in response to retinal disease and the basis for this variation is unknown. In this study, we used both bulk and single-cell RNA sequencing to compare gene expression within concentric regions of the central retina. We identified unique gene expression patterns of foveal cone photoreceptor cells, including many foveal-enriched transcription factors. In addition, we found that the genes RORB1, PPFIA1 and KCNAB2 are differentially spliced in the foveal, parafoveal and macular regions. These results provide a highly detailed spatial characterization of the retinal transcriptome and highlight unique molecular features of different retinal regions.
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