By using NGS in 254 known and candidate genes we were able to detect known and novel mutations in 57% of families tested. In order to achieve this goal, we applied a rigorous protocol (Figure ). To our knowledge, this is the first report using NGS to investigate all inherited retinal disorders at once. In a study restricted to adRP, Bowne and co-workers used a similar approach including 46 known and candidate genes for adRP [18
]. All their cases had previously been screened and excluded for most of the known genes underlying adRP. The authors were able to identify known or novel mutations in five out of 21 cases in genes not included in a pre-screening [18
]. This added five patients to their adRP cohort with known gene defects, indicating that 64% of their patients show known mutations with new genes still to be discovered in the remaining 36%. The current study provides a more exhaustive tool, since it incorporates screening of 254 genes implicated in various retinal disorders of different inheritance patterns and additional candidate genes for these phenotypes. With this approach a cohort of both pre-screened and unscreened samples, was investigated. The mutation detection rate of 57% is high and was never obtained before by high throughput screening methods. Furthermore, this approach is probably less time consuming and expensive than existing methods such as direct sequencing of all known genes or microarray analysis. Of note however is one of the variants detected with the NGS approach (i.e. p.V973L exchange in GUCY2D
), which was not confirmed by direct Sanger sequencing, suggesting the possibility of false positive using the high throughput screening. Verification by direct Sanger sequencing of most likely pathogenic variants is therefore essential to validate NGS data, although the false positive rate is assumed to be low (in our study 1/28 verified sequence variants represented a false positive).
Overall, the study of 20 subjects from 17 families by NGS showed that most of the targeted regions are well covered (more than 98%). However, some of the regions showed a lower coverage (GC-rich regions) or were not captured (repetitive regions). This was for instance the case for two genes underlying cCSNB, (i.e. NYX and GRM6) and the repetitive region of ORF15 of RPGR. For GC-rich regions the capture design could be improved in the future by modifying NGS chemistry, as it was successfully achieved for Sanger sequencing using different additives, which improved the amplification and subsequent sequencing. If repetitive regions like ORF15 of RPGR remain problematic for sequencing by NGS, direct Sanger sequencing of these targets might be the first screening of choice; in particular for disorders caused only by a few gene defects such as CSNB, and xl-RP.
By applying NGS sequencing to our retinal panel, known and novel mutations were detected in different patients. We believe that our diagnostic tool is particularly important for heterogeneous disorders like RP, for which many gene defects with different prevalence have been associated to one phenotype. It also allows the rapid detection of novel mutations in minor genes which are often not screened as a priority by direct Sanger sequencing. This was the case in our study for three individuals from one family with adRP in which NGS detected a novel PRPF8
mutation in both affected and one unaffected family member (Table , Figure ). In this family, the RP phenotype is mild and therefore it is possible that the unaffected member may develop symptoms later in life or alternatively it may be a case of incomplete penetrance as reported for another splicing factor gene, PRPF31
and recently for PRPF8
as well [19
]. Interestingly, a novel TRPM1
mutation was identified in a patient with adCSNB, a gene previously only associated with arCSNB [23
]. This is the first report of a TRPM1
mutation co-segregating with ad Schubert-Bornschein type complete CSNB. Since the location of this mutation is not different compared to other mutations leading to arCSNB, it is not quite clear how TRPM1
mutations might lead to either ad or arCSNB. Functional investigations are needed to validate the pathogenicity of this variant. Furthermore, this finding suggests that TRPM1
heterozygous mutation carriers from arCSNB families should be investigated by electroretinography to determine whether they display similar retinal dysfunction as in affected members of the presented adCSNB family. Detection of a novel RPGR
splice site mutation in family 146 presented a challenge. The actual disease causing change was concealed under a wrongly annotated rs62638633, which had previously been clinically associated to RP by a German group http://www.ncbi.nlm.nih.gov/sites/varvu?gene=6103&rs=62638633
, (personal communication, Markus Preising). These observations indicate that the stringent filtering we applied initially can mask those referenced disease causing variants. Bearing this in mind one can still first investigate unknown variants, but should then examine dbSNP for referenced variants either described to be disease causing, having a low minor allele frequency or present in interesting candidate genes. An accurate discrimination of non-pathogenic polymorphisms versus disease causing polymorphism in SNP databases is warranted to resolve this challenge.
In six families from the investigated cohort the disease causing mutations still remain to be identified. In the Stargardt patient with no pathogenic ABCA4
mutations two variants in CFH
were detected, one of which (rs1061170) had previously been reported to predispose to age related macular degeneration (AMD) [27
]. The second CFH
change is a novel variant, affecting a highly conserved residue, not found in NGS data from the other 19 samples and never associated with a disease. The variants co-segregated in the only available family members, which were the patient's parents. Apart from the association with AMD, CFH
mutations have been previously associated with renal diseases, the most common being membranoproliferative glomerulonephritis and hemolytic uremic syndrome, which can be also associated with an eye phenotype [30
]. No renal dysfunction was present in our patient. To validate if the two variants identified in CFH
are indeed disease causing, the DNA samples from other available family members for co-segregation analysis as well as characterization of functional consequences of the novel variant are needed. One patient with complete CSNB had an affected nephew and thus x-linked inheritance was assumed. However, neither Sanger nor NGS detected a mutation in the only known x-linked gene, NYX
, causing cCSNB. To exclude recessive inheritance TRPM1
were investigated in detail. Indeed the patient carried a novel heterozygous TRPM1
variant, which affects a highly conserved amino acid and was not identified in the other 19 samples investigated here (Table ). However, direct Sanger sequencing of lower covered regions did not identify a second mutation in this gene. Similarly no mutations in GRM6
were identified. These findings outline the need for additional family members to determine, through co-segregation, the pathogenicity of the numerous variants identified by NGS. This was also true for two other families with nonsense mutations in CUBN
(Fam795) and RP1L1
(Fam761) (Table ). The nonsense mutation in CUBN
, co-segregated with the phenotype in most of the family members (Figure ). Had we not had access to additional family members, we might have retained this gene defect as the underlying cause for adCD and considered CUBN
as a new gene involved in adCD. None of the other putatively pathogenic mutations identified in CUBN, TRPM1
co-segregated with the phenotype in this family (Table , Figure ). RP1L1
was already a candidate for adRP [32
] but was previously associated with occult macular dystrophy [33
]. In our study, this variant did not co-segregate with the phenotype in other affected family members (data not shown).
This NGS study ended with six genetically unresolved families, which can be further investigated with whole exome sequencing. Although, no clear information about the actual percentage of missing gene defects underlying each group of inherited retinal disorders exists, previous studies have reported that in many cases the genetic cause still needs to be determined [18
]. Whole exome sequencing approaches allow the detection of both, novel and known gene defects, but also generate numerous variants and therefore require the inclusion of more than one DNA sample for each family to rapidly exclude non-pathogenic variants. Due to the higher costs of exome sequencing for one sample compared to targeted sequencing, we propose to initially perform targeted sequencing in the index patient and proceed only after exclusion of a known gene defect to whole exome sequencing.