Coloboma and microphthalmia (C/M) are related congenital eye malformations, which can cause significant visual impairment. Molecular diagnosis is challenging as the genes associated to date with C/M account for only a small percentage of cases. Overall, the genetic cause remains unknown in up to 80% of patients. High throughput DNA sequencing technologies, including whole-exome sequencing (WES), are therefore a useful and efficient tool for genetic screening and identification of new mutations and novel genes in C/M. In this study, we analyzed the DNA of 19 patients with C/M from 15 unrelated families using singleton WES and data analysis for 307 genes of interest. We identified seven novel and one recurrent potentially disease-causing variants in CRIM1, CHD7, FAT1, PTCH1, PUF60, BRPF1, and TGFB2 in 47% of our families, three of which occurred de novo. The detection rate in patients with ocular and extraocular manifestations (67%) was higher than in patients with an isolated ocular phenotype (46%). Our study highlights the significant genetic heterogeneity in C/M cohorts and emphasizes the diagnostic power of WES for the screening of patients and families with C/M.

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK’s guidelines. Additionally, DeepVariant was complemented by GATK’s workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields.

Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, and analyze conditions that lead to ABCA1 dysregulation in induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs). Our results indicate that the risk-conferring alleles rs1883025 (C) and rs2740488 (A) in ABCA1 are associated with increased ABCA1 mRNA and protein levels and reduced efficiency of cholesterol efflux from the RPE. Hypoxia, an environmental risk factor for AMD, reduced expression of ABCA1 and increased intracellular lipid accumulation. Treatment with a liver X receptor (LXR) agonist led to an increase in ABCA1 expression and reduced lipid accumulation. Our data strengthen the homeostatic role of cholesterol efflux in the RPE and suggest that increasing cellular cholesterol export by stimulating ABCA1 expression might lessen lipid load, improving RPE survival and reducing the risk of developing AMD.

RPGR exon ORF15 variants are one of the most frequent causes for inherited retinal disorders (IRDs), in particular retinitis pigmentosa. The low sequence complexity of this mutation hotspot makes it prone to indels and challenging for sequence data analysis. Whole-exome sequencing generally fails to provide adequate coverage in this region. Therefore, complementary methods are needed to avoid false positives as well as negative results. In this study, next-generation sequencing (NGS) was used to sequence long-range PCR amplicons for an IRD cohort of African ancestry. By developing a novel secondary analysis pipeline based on de novo assembly, we were able to avoid the miscalling of variants generated by standard NGS analysis tools. We identified pathogenic variants in 11 patients (13% of the cohort), two of which have not been reported previously. We provide a novel and alternative end-to-end secondary analysis pipeline for targeted NGS of ORF15 that is less prone to false positive and negative variant calls.

Targeted resequencing technologies have allowed for efficient and cost-effective detection of genomic variants in specific regions of interest. Although capture sequencing has been primarily used for investigating single nucleotide variants and indels, it has the potential to elucidate a broader spectrum of genetic variation, including copy number variants (CNVs). Various methods exist for detecting CNV in whole-genome and exome sequencing datasets. However, no algorithms have been specifically designed for contiguous target sequencing, despite its increasing importance in clinical and research applications. We have developed cnvCapSeq, a novel method for accurate and sensitive CNV discovery and genotyping in long-range targeted resequencing. cnvCapSeq was benchmarked using a simulated contiguous capture sequencing dataset comprising 21 genomic loci of various lengths. cnvCapSeq was shown to outperform the best existing exome CNV method by a wide margin both in terms of sensitivity (92.0 versus 48.3%) and specificity (99.8 versus 70.5%). We also applied cnvCapSeq to a real capture sequencing cohort comprising a contiguous 358 kb region that contains the Complement Factor H gene cluster. In this dataset, cnvCapSeq identified 41 samples with CNV, including two with duplications, with a genotyping accuracy of 99%, as ascertained by quantitative real-time PCR.