Mutations in CRB1 are a common cause of congenital or severe childhood-onset RD accounting for up to 10.1% of LCA/EORD patients and 2.7% in RP cases, as described in a recent meta-analysis of CRB1 mutations . The above frequencies may be underestimated, as sequencing of entire coding regions had not been systematic performed. In this sense, CRB1 analysis in large cohorts of patients has been mostly performed by us and other groups using SSCP or chip-based screening for known RD mutations [3–5, 8, 10, 15, 16, 21]. Only Coppieters et als performed a subsequent an exhaustive screening of CRB1 and other LCA-related genes by Sanger sequencing in all patients with negative chip results obtaining a higher frequency (16%) of CRB1 mutations . Similarly, we aimed to evaluate herein the real relevance of CRB1 mutations in the Spanish population using an additional comprehensive mutational screening by HRM and Sanger sequencing, identifying causative variants in 11% of LCA/EORP cases. It is noteworthy that we found a high number of private CRB1 mutations in our cohort confirming the usefulness of in-depth CRB1 genetic analysis to identified novel variants undetectable by genotyping microarrays.
Hitherto, few reports have focused on the implication of CRB1 mutations in RP patients . CRB1 defects seemed to explain <3% of EORP cases as described in our previous reports in Spanish patients [15, 16] and other population [8, 10]. However, it is remarkable that herein up to 9% of our EORP patients carried two CRB1 mutations. We presumed that this improved detection rate of CRB1 variants could be due not only to the use of an updated arRP microarray containing some mutations first described in the Spanish population but also for carrying out a comprehensive analysis of the whole gene. Although the most frequent mutation in EORP patients is p.Cys948Tyr occurring worldwide , it is noteworthy that 25% of pathogenic CRB1 alleles correspond to 2 apparently population-specific mutations, the c.498_506del and p.Ile1100Thr variants . In fact, 19 of 25 different EORP-associated CRB1 mutations, representing 61% of CRB1 alleles, were first identified in our cohort, that evidences a high frequency of population-specific mutations in the Spanish EORP patients.
Focusing on the LCA patients, CRB1 defects accounted for 14% of Spanish cases. In view of the high detection rate obtained, APEX genotyping seems to be a quite effective and fast diagnostic approach to detect CRB1 mutations in our LCA cohort. However, 4 additional patients were identified carrying novel mutations by a further CRB1 complete screening. The frequency of CRB1 mutations varies among different studies, ranging from 1% in a cohort of LCA patients hailing from worldwide countries  to 16% in Belgium . Although the prevalence of CRB1 defects is apparently identical between the Belgian and Spanish cohorts, substantial differences are evidenced in the mutational CRB1 spectrum. The missense variant p.Cys948Tyr is also the most frequent disease allele in Spanish LCA patients, representing 31% of total alleles, but not in Belgium being less prevalent with a frequency of 23% . In contrast, the most recurrent allele in Belgian patients, p.Lys801*, was only detected in one Spanish LCA case. Interestingly, 12 out 17 different LCA-associated CRB1 variants, representing 44% of total alleles, seem to be specific to our cohort.
Mutations in CRB1 were previously associated with a wide range of phenotypic manifestations [1–6, 23]. Complete loss of function of the CRB1 protein seems to be more related with development of LCA phenotype, while some residual functionality may remain in childhood-onset patients . Nevertheless, a clear genotype–phenotype correlation could not be established since 2 seeming LOF alleles were not only identified in LCA but also EORD in several cohorts . Interestingly, we observed that a combination of 2 null mutations was only found in about 40% of LCA patients but in none of our EORP patients, as showed in Additional file 2: Figure S6. Null alleles were significantly more frequent in LCA that in EORP patients (χ2 = 10.2, p<0.001), as previously suggested . However, a similar proportion of LCA and EORP cases carried a combination of null-allele with missense mutation and several LCA patients also presented 2 missense alleles, being p.Cys948Tyr always involved. In consequence, it is not easy to assign a specific allele combination to a particular phenotype, suggesting a strong influence of environmental factors or genetic modifiers on the severity of the disease. In fact, it is likely that the new next-generation sequencing (NGS) technology will help to identify potential genetic variants that modulate LCA and EORP phenotypes associated to CRB1 defects.
In view of the high frequency of compound heterozygous variants in CRB1, we evaluated the HRM technology that is considered a powerful approach to efficiently discriminate heterozygous variants , as an alternative to Sanger sequencing. We accurately identified heterozygous variants in all abnormal melting curves (specificity of 100%); however, we also found 4 false negatives thus obtaining a lower sensitivity of 73% for CRB1 screening by HRM. Several factors such as amplicon size or GC content could influence the sensitivity to detect melting variants and explain the unexpected high number of false negatives in this analysis as opposed to previously reports .
This work supports the importance of comprehensive genetic studies in order to ascertain the real prevalence of retinal gene defects in large cohorts of well-clinically phenotyped patients. Main limitations to in-depth genetic analysis of RD are related to the use of expensive and time-consuming techniques. However the recent advances in NGS technologies and their progressive implementation in the clinical diagnosis will help to improve the molecular diagnosis and also to shed light on genetics of retinal dystrophies.