Introduction

Retinal dystrophies (RD), comprising the wide spectrum of retinal degeneration, are rare genetic conditions leading to visual deficiency and in some instances to blindness [1]. These diseases affect roughly one person out of 4,000 and are the result of the progressive loss or dysfunction of photoreceptors, the light-sensing cells of the eye [2]. RD are caused by mutations in more than 100 different genes (RetNet; https://sph.uth.edu/Retnet/home.htm). Mutations causing RD can occur in any of these genes, as well as in other disease genes that still await identification, and can be transmitted as dominant, recessive, or X-linked alleles. This very elevated genetic heterogeneity is possibly the highest detected so far among all Mendelian diseases and leads to a very high carrier frequency of heterozygous mutant alleles, i.e. possibly more than 1 in 4–5 individuals [3]. Molecular diagnosis is therefore an extremely daunting task since unrelated patients with the similar clinical presentations are likely to have defects in different genes, each of which with a small chance to be found positive for mutations. In other cases, accurate molecular diagnosis is hampered by limited clinical information, which is critical for efficiently deciding which genes have to be analyzed. To bypass the drawbacks of classical exon-by-exon PCR screenings, associated with very long processing times, the use of mutation- or gene-specific microarrays has been recently adopted [4]–[12], with different results reported by us and others to be largely dependent on the characteristics of the population screened [13]–[19].

Whole exome sequencing (WES) is a procedure that allows the purification by sequence capture of all exonic regions of a genome and their further processing by next-generation sequencing (NGS) [20]. Since the selection of the target regions to be sequenced by WES spans all of the genome, this method is not conventionally used to interrogate a specific DNA fragment or a limited number of genes, but conversely it is adopted to investigate genetic features at a genome scale. Moreover, in virtue of their general applicability, WES protocols and kits have diffused rapidly and have become more and more affordable, to the point of being in some instances less expensive than targeted DNA capture/NGS projects interrogating a lower number of DNA features.

In this work, we evaluate the possibility of using WES as a tool for routine molecular diagnoses in patients with apparently recessive RD. Although we specifically selected families with multiple affected members to allow validation of the findings, we willingly ignored the information related to the pedigree, to simulate the use of this technique for the large majority of people with RD, i.e. isolate patients.

Materials and Methods

Results

Detection of Mutations in Known RD Genes

Ten of the 12 index patients analyzed by WES were found to be either homozygous or compound heterozygous for variants in known RD genes that would satisfy our filtering criteria (Table 1, Figure 1).

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Figure 1. Pedigrees of patients analyzed and mutations identified in this work.

The family ID is given above the pedigree, while the individuals’ IDs are indicated below the symbols depicting them. Red circles indicate individuals whose DNA underwent WES analysis. The name of the RD gene identified as causative of the disease is given in blue. M/M, homozygous mutation; M/m compound heterozygous mutations.

https://doi.org/10.1371/journal.pone.0065574.g001

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Table 1. RD mutations identified by WES analyses.

https://doi.org/10.1371/journal.pone.0065574.t001

More specifically, 3 patients/families were positive for mutations in ABCA4, 2 had mutations in the RP1 gene, and 2 others in the CNGB3 gene. The remainder of these carried variants in CHM, USH2A, and NMNAT1. All of these mutations cosegregated perfectly with the disease in all families, according to a recessive or, in the case of family RP-1164, X-linked pattern of inheritance, as ascertained by exon-PCR and Sanger sequencing (Figure 1). Of the 15 different mutations identified, 8 were never described previously and included 7 changes that were clearly deleterious alleles (frameshifts or nonsenses, Table 1). The remaining mutation was a missense change (c.12574C>T, p.R4192C) identified in the USH2A gene. This was considered pathogenic in conjunction with another clear-cut mutation (c.920_923dupGCCA, p.H308Qfs*16) based on the following reasons. First, cosegregation analysis in the mother and 5 other siblings (2 affected and 3 unaffected) was statistically significant, with a p-value of 0.006 (Figure 1). Second, the mutation was not found in 100 ethnically-matched healthy controls or any other public database, including the one from the 1000 Genomes Project. Lastly, in silico assessment of the consequence of the missense mutation predicted the change to affect the function of the translated protein (SIFT and Polyphen).

All patients identified with disease-associated mutations had no other genes/variants that fulfilled our filtering criteria, except for individual 04/0834. This patient carried the previously-reported homozygous CNGB3 mutation p.T383Ifs*13 [31] but was also found to have two novel missense variations in the USH2A gene. Sanger sequencing failed to detect one of such USH2A variants (thus representing a false negative result) while the presence of the homozygous mutation in CNGB3 was confirmed.

The remaining 2 index patients from families RP-0886 and RP-0461 were not identified with pathogenic RD mutations. They were therefore all considered as patients for whom molecular diagnosis could not be achieved in absence of additional information.

Discussion

Because of the elevated genetic and allelic heterogeneity displayed by hereditary retinal degenerations, molecular diagnoses are in general a rather complicated task. Although precise clinical information or family history can facilitate this procedure, the number of known mutations that medical geneticists have to consider in the screening process is likely in the range of a few thousands, over more than 100 different genes. For example, approximately 600 variants/mutations in the USH2A gene alone have been reported so far (the USH2A mutations database: http://www.lovd.nl/USH2A). Furthermore, novel RD mutations are constantly being discovered, reducing the value of semi-automated analyses interrogating a specific set of DNA variants.

Starting from the evidence that costs of whole-exome sequencing are constantly lowering and that this technique, although not too sensitive, allows an unsupervised analysis of all of the coding sequences of the human genome, we reasoned that WES could in fact be used in routine DNA-based diagnosis of RD. Our results show an 83% success rate, over 12 families with seemingly recessive RD. This high success rate can partially be explained by the nature itself of recessive conditions, for which two mutations have to be present in the same gene to cause disease. This event alone enables a drastic reduction of the noise associated with WES, since the likelihood that two false negative results (e.g. real DNA changes with no pathogenic effects or simple sequencing errors) affect by chance the same gene is very low. In dominantly inherited cases, for which such a filter cannot be applied, there would be a priori no such a way of distinguishing pathogenic variants form rare DNA changes or sequencing false calls. However, although with lower efficiency, WES could in principle still be applied. Important success factors in dominant investigations would be represented by the detection of clear-cut mutations, such as indels and nonsense variants, in known disease genes and the use of control population data analyzed with WES, in order to systematically subtract the noise and enhance the signal throughout the genome. Additionally, the identification of less striking but previously-reported pathogenic changes would also suffice to provide molecular diagnoses to dominant RD cases. For instance, we could efficiently detect a novel frameshift mutation in the CHM gene in a male patient with choroideremia. Since CHM is located on the chromosome X, this patient and his affected brother do in fact carry a single causative mutation, as in individuals affected by autosomal dominant conditions. Isolated RD cases would be, in the vast majority of the circumstances, either unrecognized recessive cases or de novo dominant cases, and therefore could be analyzed by following the same procedures.

NGS has been applied previously to screen for specific forms of RD, with variable results [32]–[34]. For example, NGS provided genetic diagnosis in 36% (36 out of 100) of patients with retinitis pigmentosa [33]. After correcting for the mutation detection rate of NGS and by taking into consideration previously solved cases, the diagnostic yield increased to ∼50%. The comparatively favorable results of the current study that identified 83% of the causes of RD may be attributable to a few specific elements, including the size of the cohort analyzed, the methods and extensiveness of the genetic screenings carried out in the past, the clinical phenotype of the patients, and/or the sequencing/mapping methods of NGS itself.

Another element of note is that out of the 15 mutations identified, 8 (∼50%) were novel, despite the genes in which they were identified were extensively screened for mutations in other cohorts of patients. This finding confirms, unfortunately, the limited value of mutation-based microarrays or sequencing procedures (such as pyrosequencing) in the context of genetics of RD. We also found that wide phenotypic variability of RD and limited clinical information could preclude accurate clinical diagnosis that is essential for current targeted screening methods. In this context, the “screen all RD genes” approach has certainly benefitted three of our cases, initially diagnosed as undefined RD (patient 10/1367), LCA (patient 04/0834), or RP (patient 07/0360), but having mutations in CNGB3 and CHM causing achromatopsia and choroideremia, respectively, that would be missed by ARRP or LCA genotyping microarray. Following such findings, these patients were re-evaluated clinically, and confirmed to have achromatopsia and choroideremia.

Despite the power of WES, validation by Sanger sequencing and, if possible, of additional genetic investigation in family members of the patient remain key elements for the success of this screening technique. The value of familial analyses is particularly evident when we consider the two families for which a DNA diagnosis could not be reached. In these pedigrees, the number of candidate mutations in non-reported RD genes was simply too elevated and, in absence of an additional filtering based on co-segregation of disease and DNA changes in affected relatives, could not be reduced. Moreover, due to limitations that are intrinsic to the exome sequencing procedure, our analyses were underpowered to score DNA copy number variations (CNVs). It is therefore possible that these unsolved cases may in fact carry pathogenic CNVs in RD genes that we were unable to detect.

Methodologically, the procedures used were in general rather simple. We outsourced the sequencing to a large sequencing company, which provided us not only with the raw sequencing reads, but also with an annotated list of variants. These services represent nowadays a standard offer of many private sequencing centers as well as of internal genetic facilities, since most WES protocols and downstream analyses can be streamlined to what closely resembles routine procedures. Computer-based data analysis was also not particularly complicated; we mostly used simple text-parsing scripts and/or commercial spreadsheet programs with filtering capabilities, on common desktop computers.

In our screening we could also gain a few elements of information concerning the molecular pathology of RD mutations. Specifically, we could confirm that missense mutations in USH2A, a gene found to be mutated in patients suffering from the blindness-deafness disease known as Usher syndrome, cause in general retinitis pigmentosa without hearing loss (patient 08–177) [35]. We also confirmed that RP1, a gene that has been for a long time associated with dominant retinitis pigmentosa, can in fact carry recessive mutations, as previously reported [22], [36]–[42].

In summary, in our work we show that whole-exome sequencing could represent a useful tool for molecular diagnosis of hereditary retinal degeneration, a disease for which standard screening procedures still struggle to produce information in a cost- or time-efficient manner. Further reduction in prices and improvement in sensitivity and specificity, already in progress, would probably make of WES the technique of choice for all future routine DNA diagnoses.

Supporting Information

Acknowledgments

We would like to acknowledge the Spanish families of patients for taking part in this research and Dr. Carlos Vazquez, Miguel Ángel López, Paola Benaglio and M^a Belen Benavides for their contributions to this work.