Novel RP1 mutations and a recurrent BBS1variant explain the co-existence of two distinct retinal phenotypes in the same pedigree
© Méndez-Vidal et al.; licensee BioMed Central Ltd. 2014
Received: 10 June 2014
Accepted: 3 December 2014
Published: 14 December 2014
Molecular diagnosis of Inherited Retinal Dystrophies (IRD) has long been challenging due to the extensive clinical and genetic heterogeneity present in this group of disorders. Here, we describe the clinical application of an integrated next-generation sequencing approach to determine the underlying genetic defects in a Spanish family with a provisional clinical diagnosis of autosomal recessive Retinitis Pigmentosa (arRP).
Exome sequencing of the index patient resulted in the identification of the homozygous BBS1 p.M390R mutation. Sanger sequencing of additional members of the family showed lack of co-segregation of the p.M390R variant in some individuals. Clinical reanalysis indicated co-ocurrence of two different phenotypes in the same family: Bardet-Biedl syndrome in the individual harboring the BBS1 mutation and non-syndromic arRP in extended family members. To identify possible causative mutations underlying arRP, we conducted disease-targeted gene sequencing using a panel of 26 IRD genes. The in-house custom panel was validated using 18 DNA samples known to harbor mutations in relevant genes. All variants were redetected, indicating a high mutation detection rate. This approach allowed the identification of two novel heterozygous null mutations in RP1 (c.4582_4585delATCA; p.I1528Vfs*10 and c.5962dupA; p.I1988Nfs*3) which co-segregated with the disease in arRP patients. Additionally, a mutational screening in 96 patients of our cohort with genetically unresolved IRD revealed the presence of the c.5962dupA mutation in one unrelated family.
The combination of molecular findings for RP1 and BBS1 genes through exome and gene panel sequencing enabled us to explain the co-existence of two different retinal phenotypes in a family. The identification of two novel variants in RP1 suggests that the use of panels containing the prevalent genes of a particular population, together with an optimized data analysis pipeline, is an efficient and cost-effective approach that can be reliably implemented into the routine diagnostic process of diverse inherited retinal disorders. Moreover, the identification of these novel variants in two unrelated families supports the relatively high prevalence of RP1 mutations in Spanish population and the role of private mutations for commonly mutated genes, while extending the mutational spectrum of RP1.
KeywordsBardet-Biedl syndrome BBS1 Inherited retinal dystrophies Next-generation sequencing Retinitis pigmentosa RP1
Genetic testing of inherited retinal dystrophies (IRDs) is a highly complex and time-consuming process. More than 200 genes have been associated with this heterogeneous group of disorders (RetNet, https://sph.uth.edu/retnet/home.htm), characterized by the primary dysfunction or loss of photoreceptors leading to progressive visual impairment and blindness. Further complicating the molecular diagnosis of IRDs, clinical diagnosis can also be challenging due to IRD clinical features overlap. The identification of causative mutations is important for several reasons, among them, it allows geneticists to give an adequate genetic counseling to the families, to establish genotype-phenotype correlations that predictably improve the prognosis of the disease, and to monitor the efficacy and safety of new therapeutic options.
Retinitis Pigmentosa (RP; MIM #268000) represents the most prevalent clinical subtype of IRDs, affecting 1 in ~4,000 individuals. The clinical course of RP involves progressive degeneration of rods and subsequent involvement of cones as the disease progresses. To date, more than 60 genes have been associated with RP, being EYS the most prevalent gene worldwide –. Although RP is usually manifested as a non-syndromic disease, it can also be associated with a wide variety of extraocular symptoms constituting syndromic forms of RP, being Bardet-Biedl syndrome one of the most common (BBS; MIM #209900). BBS is a model ciliopathy with high genetic and clinical heterogeneity, generally inherited as an autosomal recessive trait . The estimated prevalence in Europe is approximately 1/125,000 . This disorder is characterized by a combination of clinical symptoms including obesity, RP, post-axial polydactyly, polycystic kidneys and learning disabilities, some of which may appear many years after the onset of the disease. Clinical expression is variable but many patients manifest most of the clinical symptoms during the course of illness. The number of causative genes is relatively high with 18 different genes associated so far (Ret Net; accessed 2014 Mar 1), including BBS1 (MIM #209901), that accounts for approximately 40 % of BBS families.
One of the first RP-associated genes to be discovered was RP1 (MIM #603937). This gene is located on chromosome 8q12 and consists of 4 exons . It encodes an oxygen-regulated photoreceptor protein with two microtubule-binding, doublecortin-like domains (DCX) at the N-terminus involved in microtubule stabilization. Additionally, the C-terminal region has been implicated in the binding of RP1 to proteins destined for the outer segment . Human RP1 specifically localizes to the connecting cilia of rod and cone photoreceptors also suggesting a role in the transport of proteins between the inner and outer segments and/or in maintenance of cilial structure . The vast majority of pathogenic mutations in this gene are truncating variants clustered in exon 4 (Human Gene Mutation Database, HGMD, http://www.hgmd.cf.ac.uk/ac/index.php; accessed 2014 Mar 24). Mutations in RP1 can cause both autosomal dominant (adRP) , or autosomal recessive RP (arRP) –, and although several mechanisms have been suggested to explain the mutational mechanisms in RP1 leading to dominant or recessive RP , no clear models have been proposed.
The recently introduced next-generation sequencing (NGS) technologies have greatly facilitated the molecular diagnosis of IRD, in particular, in poorly defined or misclassified clinical cases . At present, targeted analysis of disease-specific candidate genes is most suitable for diagnostic applications as it facilitates functional interpretation of sequence variations and overcomes limitations in computational analysis . In this study, we describe the successful application of an integrated NGS strategy to provide an accurate clinical and molecular diagnosis of concurrent BBS and arRP in a family provisionally diagnosed of arRP. The definitive clinical diagnosis of the index patient was made based on clinical re-examination and sequencing data generated through whole-exome sequencing to identify the BBS1 p.M390R mutation  responsible for the BBS phenotype. Likewise, disease-targeted gene sequencing resulted in the identification of two novel truncating mutations in RP1 (p.I1528Vfs*10 and p.I1988Nfs*) in extended family members affected of arRP.
Subjects and clinical assessment
IRD panel test samples
c.2843G > A
c.10712C > T
c.2276G > T
c.1297-2A > G
c.301C > T
c.2029C > T
c.1481C > T
c.3386G > T
c.932G > A
c.701G > A
c.1025C > T
c.646C > T
c.626C > A
c.544G > A
c.769C > T
DNA samples from individuals III:1, III:2, IV:1, and IV:2 were processed for WES while DNA samples from 18 patients with previously identified mutations and from individual II:5 were processed for gene panel sequencing.
Previous molecular genetic analysis
The index patient was first analyzed and excluded for known mutations in arRP genes by applying commercially available microarray analysis (Asper Biotech, Tartu, Estonia), a custom genome resequencing microarray  and by direct sequencing of EYS . Causative mutations of the IRD panel validation samples were previously identified by different strategies (Table 1).
Whole-exome sequencing and data analysis
Library preparation and exome capture were performed using NimbleGen SeqCap EZ Exome libraries V3 (Roche NimbleGen, Inc., Madison, WI, USA) and sequenced on a SOLiD 5500xl platform. These procedures and the analysis of data from deep sequencing were carried out as previously described ,.
Design of the capture IRD panel
List of genes included in the capture IRD panel
NCBI reference sequence
ADRP, ARRP, ARCRD, ARMD, STGD
USH, Deafness alone or syndromic
USH, Deafness alone or syndromic
ADRP, ARRP, ARESCS
ARRP, ADCRD, ADMD
ADRP, ADMD, ADCRD and digenic
ADRP, ARRP, ADCSNB
XLRP, XLCRD, XLMD
DNA library preparation and targeted sequencing
Library preparation was performed according to the manufacturer’s protocol (GS FLX Titanium Rapid Library Preparation Method Manual, January 2010 version). Briefly, 500 ng of input genomic DNA was fragmented by nebulization resulting in a majority of fragments between 600-900 bp. Fragments were end repaired, ligated to the provided adaptors and eluted in water following small fragments removal using AMPure Beads (Beckman Coulter, Agencourt, Beverly, MA). The library was amplified for 12 cycles by pre-capture ligation-mediated PCR (LM-PCR) using FastStart High Fidelity PCR System (Roche, Basel, Switzerland) and specific primers for the adaptors. After purification, 1 μg of LM-PCR product was hybridized to the custom designed SeqCap EZ Library of biotinylated probes for 72 h at 47°C. Captured DNA fragments were purified with streptavidin-conjugated magnetic beads (Invitrogen, Carlsbad, CA, USA) and washed. Amplification was performed for 15 cycles by post-capture LM-PCR using FastStart High Fidelity PCR System. The final concentration of each captured library was measured with Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA) and diluted to 106 molecules/μl. Emulsion PCR was performed according to the manufacturer’s instructions (emPCR Amplification Method Manual-Lib-L, GS Junior Titanium Series, March 2012). To perform the emPCR an input of 0.7 molecules per bead was chosen. After enrichment, about 500,000 beads were sequenced on 454 Roche GS Junior sequencer according to the manufacturer’s protocol (Sequencing Method Manual GS Junior, Titanium Series, March 2012).
Verification and determination of the pathogenicity of variants
Predicted disease-causing variants were confirmed by Sanger sequencing using specific primers designed by Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) and co-segregated in available family members DNA samples. Novel variants were subsequently screened in 200 healthy matched control subjects by Sanger sequencing and in Exome Variant Server (http://evs.gs.washington.edu/EVS/). The pathogenicity of novel missense substitutions was evaluated using Polymorphism Phenotyping v2 (PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/) and Sorting Intolerant From Tolerant (SIFT, (http://sift.bii.a-star.edu.sg) scores. The correct name of the variation according to the Human Genome Variation Society (http://www.hgvs.org) nomenclature guidelines was checked using Mutalyzer (http://www.LOVD.nl/mutalyzer).
Microsatellite marker analysis
Microsatellite marker analysis was carried out in available family members DNA samples of the arRP branch. For this purpose, a total of five microsatellite markers flanking RP1 were selected from UCSC Genome Browser. PCR amplification using genomic DNA samples and primers closely flanking the region containing microsatellite repeats were used for analysis (Additional file 1). PCR products were genotyped using an ABI-3730 sequencer (Applied Biosystems, USA), and analyzed by GeneMapper v.4.0 software (Applied Biosystems).
Mutational screening of RP1in additional IRD patients
To detect additional cases carrying the novel RP1 mutations, we performed Sanger sequencing in 96 IRD patients of our cohort without a genetic diagnosis. This study group consisted of 75 cases clinically diagnosed of arRP, 3 of adRP, 3 of cone-rod dystrophies, 6 of Leber Congenital Amaurosis, and 9 of Stargardt disease.
WES and clinical refinements
The index patient (IV:2) (Figure 1) had received a provisional diagnosis of arRP based on previous family history (II:1, II:5, II:9 and III:8) with clinical manifestations typical of a severe form of RP characterized by early age of onset and a rapid progression of the disease, leading to a total blindness before age 30 in most patients. The first symptom of the index patient was night blindness at age of six, followed by decreased visual acuity, reduced visual field, dyschromatopsia and photophobia. The fundus examination showed signs of RP such as bone spicule pigmentation and attenuation of the retinal vessels.
In order to identify causal mutations, we sequenced the exomes of the index patient, one healthy sibling (IV:1) and both unaffected parents (III:1 and III:2). This allowed the identification of the known homozygous p.M390R mutation in the BBS-associated gene BBS1, previously reported by Mykytyn et al, . Lack of segregation of this variant in extended family members prompted us to clinically reevaluate the entire family focusing on the identification of extraocular symptoms associated with BBS. We found that the index patient showed characteristic BBS features including obesity, learning disabilities and postaxial polydactyly, resulting in a clinical reclassification of this family branch from arRP to BBS. In contrast, fundus examination in the rest of affected members showed typical signs of RP with pale optic nerve disc, retinal vessels attenuation and bone spicule pigmentation in the periphery, confirming a diagnosis of non-syndromic arRP in individuals II:5, II:9 and III:8. Therefore, the causal mutation of this family branch remained to be discovered.
Given that individual II:1 was also affected of arRP, we hypothesized that patient III:1 must be an obligate carrier of one of the arRP mutations. To assess this possibility, a screening of heterozygous mutations in known RP genes present in the WES data of individual III:1 was carried out as a first approach to detect the cause of the disease in the arRP members, but no candidates were found.
Validation of the customized IRD panel
The genetic cause of arRP was studied by sequencing 26 IRD genes. In order to assess the reliability of our capture panel as a diagnostic tool, we tested 18 samples with known causative variants on exons or bordering sequences in 17 retinal disease genes included in the panel. A total of 21 mutations were checked, 6 indels and 15 SNVs.
Detection, filtering, and verification of variants
On average, targeted sequencing of validation samples revealed 2,318 variants per patient (616 SNVs and 1,702 indels). The filtering of variants was performed as detailed in Materials and Methods and resulted in a mean of 48 variants per sample. After comparing with human mutation databases HGMD and ClinVar, a mean of 2 reported variants were identified for each sample, among which the causative mutation was found in 14 cases. The prioritizing step left 1-2 candidate variants per sample to be validated. After validation, 22 variants were considered possible causative mutations, of which 21 matched the causal mutations previously identified, and only one additional novel mutation in RP1 in sample #8 was found (c.3956A > G, p.E1319G). Therefore, all variants were properly redetected using this NGS approach and verification of underrepresented NGS regions by Sanger sequencing was not required in this study.
Identification of pathogenic mutations
To discard the possibility that the two mutations were located on the same allele, we performed a linkage analysis using five microsatellite markers flanking the RP1 gene on individuals II:5, II:7, II:9 y III:8. The haplotypes confirmed that each mutation is found in different alleles (Additional file 2).
Mutational screening of RP1mutations in unresolved IRD cases
Direct full sequencing of the two novel RP1 mutations in 96 IRD patients without a molecular diagnosis allowed the detection of the c.5962dupA mutation in homozygosity in one additional family of our cohort (Additional file 3). This family was not related to family RP368. Clinically, patient with homozygous mutation c.5962dupA showed signs of typical early onset RP and resembles arRP affected members of family RP368.
Clinical and genetic complexity of IRD makes the accurate diagnosis of some cases only accessible by NGS approaches. Here, we followed a strategy that combines exome and disease-targeted gene panel sequencing to assist in the molecular diagnosis of a family affected of both BBS and RP. Whole exome sequencing allowed the identification of the BBS1 p.M390R mutation as the BBS-causing variant while the genetic cause of non-syndromic arRP was assessed by gene panel sequencing.
Compared to exome sequencing, targeting fewer genes has clear advantages including the analysis of more patient samples per instrument cycle, a greater depth of coverage, a simplified data interpretation and less restrictive ethics. Validation of our disease-targeted gene panel was critical to ensure high quality interpretation of clinical evidences. All types of variants were detected by this platform indicating a high specificity of mutation detection. Of note, the identification of the c.2405_2406delAG variant located in the hot spot exon ORF15 of RPGR, a domain that usually remains problematic for sequencing, indicated that the enrichment technology produced suitable calls for use in clinical laboratories.
Analysis of our NGS data also resulted in the identification of definite candidate variants for each test sample with the exception of validation sample #8, harboring not only the causal adRP mutation p.R677* in RP1, but also an additional variant (p.E1319G). The fact that the vast majority of pathogenic mutations in RP1 result in premature stop codons (nonsense and frameshift mutations) indicate that the most probable adRP-causative mutations in validation sample #8 is p.R667* and not p.E1319G, which might be secondary to the disease and simply reflect the high frequency of carriers harboring heterozygous mutant alleles of IRD genes in the general population . Additionally, the p.R677* mutation has been reported as the most frequently pathogenic RP1 mutation , with a location consistent with a dominant inheritance pattern .
Studies performed after this panel design have shown that, of all solved cases in our cohort, 98% of families carry mutations in one of the genes included in the panel while 2% of cases harbor mutations in other genes (unpublished data), indicating that, as expected, we primarily find novel mutations in prevalent genes in our population. Therefore, although we did not aimed at evaluating the diagnostic yield in this study, we can expect that a large proportion of pending cases may be solved using this panel which is remarkable given the small target size compared to other IRD panels ,–. Nonetheless, we are aware that to increase the diagnostic yield of the panel, the analysis of other types of variants such as large CNVs or structural variants must be considered . Also, genes newly found to be mutated in our cohort as well as non-coding regions with previously reported deep-intronic pathogenic mutations – must be included.
The implementation of this tool for the molecular diagnosis of arRP individuals resulted in the reliable identification of two novel RP1 mutations (c.4582_4585delATCA; p.Ile1528Valfs*10 and c.5962dupA; p.Ile1988Asnfs*3). Family segregation showed indeed the presence of the variant c.5962dupA in heterozygous state in individual III:1. However, this variant was filtered out during the re-analysis of the WES data of heterozygous variants in RP genes in this individual due to low coverage (4x), supporting the use of gene panels to increase the coverage of certain target regions.
Screening of our cohort for these mutations led to the molecular diagnosis of an unrelated arRP family carrying the c.5962dupA variant in homozygosis. The presence of this variant in two unrelated families of our cohort seems to indicate the recurrent nature of this novel variant in Spanish population. These data are in agreement with a recent report showing that the contribution of RP1 to arRP in the Spanish population may have been underestimated .
It has been previously hypothesized that the specific location of mutations in RP1 may be an important factor in determining their functional effect . Given that both frameshift mutations found in this study are located in the second half of exon 4, the mutated mRNA would not be theoretically susceptible to the nonsense-mediated decay (NMD) pathway , resulting in prematurely truncated proteins. It has been proposed that while larger disruptions of RP1 are often associated with adRP due to deleterious effects of the truncated proteins, the lack of only the most terminal portion results in a loss of RP1 function, causing arRP ,. This would explain that the two novel detected mutations are pathogenic in compound heterozygosity or homozygosity, while heterozygous carriers of only one of them are asymptomatic. Besides their location, the type of mutation appears to be also important to the etiopathogenesis of RP associated with RP1 mutations. Both families diagnosed in this study showed similar clinical features with severe RP, characterized by early age of onset and a rapid progression of the disease. These clinical characteristics are consistent with previous studies , describing that patients with arRP due to RP1 mutations show severe RP and present equivalent phenotypes in different families.
Previous reports have shown other cases of apparent clinical heterogeneity due to independent segregation of two different retinal phenotypes secondary to mutations in two different genes ,. Interestingly, other cases of concurrent retinal disorders have been explained by the presence of p.M390R and mutations in the arRP-associated genes C2ORF71 and RP1 ,. In this study we further highlight how isolated population or consanguinity could increase the risk factor of developing diverse IRDs in the same family. Consequently, it is important to consider the existence of different genetic causes in the same family when clinical manifestations and genetic data do not correlate.
The genetic findings for RP1 and BBS1 genes revealed co-occurrence of two distinct retinal phenotypes resulting in the accurate diagnosis of a poorly defined clinical case. The detection of two novel pathogenic RP1 variants p.Ile1528Valfs*10 and p.Ile1988Asnfs*3 suggests that the use of IRD panels of a small size, limited to prevalent genes of a particular population, together with an optimized data analysis pipeline, is an efficient and cost-effective approach for the diagnosis of diverse retinal disorders. Moreover, the implementation of this panel as a diagnostic tool can be useful to determine the prevalence of known IRD genes in our cohort. In fact, the identification of RP1 mutations in two unrelated families supports the relatively high prevalence of this gene in Spanish population.
The authors are grateful to the families described in this study. This work was supported by Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, Spain (PI1102923, CIBERER ACCI and CDTI FEDER-Innterconecta EXP00052887/ITC-20111037), and the Foundation Ramón Areces (CIVP16A1856). The CIBERER is an initiative of the ISCIII, Spanish Ministry of Economy and Competitiveness. NB-G is supported by fellowship FI12/00545 from ISCIII.
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