Mutations in RAB28, Encoding a Farnesylated Small GTPase, Are Associated with Autosomal-Recessive Cone-Rod Dystrophy

Main Text

Cone-rod dystrophy (CRD [MIM 120970]) is characterized by primary loss of cone photoreceptors and subsequent or simultaneous loss of rod photoreceptors. The symptoms include poor visual acuity, disturbances in color vision, decreased sensitivity of the central visual field, and photo aversion. Because rods are also involved, impairment of night vision and peripheral vision loss can occur. The fundus appearance can vary from normal to a bull’s eye maculopathy, or to marked atrophy of the macular region, with a variable degree of temporal pallor of the optic disc.¹ Visual fields display a central scotoma and full-field electroretinography (ERG) records a progressive deterioration of cone-derived amplitude responses, which are more reduced than, or equally reduced as, rod-derived responses.

CRD has an estimated prevalence of 1:30,000 to 1:40,000^1–3 and displays all types of Mendelian inheritance. Seven genes have been implicated in the autosomal recessive (ar) form, the most prevalent mode of inheritance, i.e., ABCA4 (MIM 601691),⁴ ADAM9 (MIM 602713),⁵ C8orf37 (MIM 614477),⁶ CERKL (MIM 608381),⁷ EYS (MIM 612424),^8,9 RPGRIP1 (MIM 605446),¹⁰ and TULP1 (MIM 602280).¹¹ However, mutations in these genes explain only a minority of the cases.¹²

The goal in this study was to identify genes underlying arCRD by using a combined approach of homozygosity mapping and exome sequencing. Such an approach has proven to be very effective in the discovery of genetic defects in several rare autosomal recessive diseases.^13–16 This study was approved by the local medical ethics committees and adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent prior to participation in the study.

To localize the genetic defect in a German family with three siblings affected by CRD (family A; Figure 1), the DNA of all three affected individuals was analyzed by using Affymetrix GeneChip Human Mapping 250K SNP arrays (Affymetrix, Santa Clara, CA, USA), and regions of homozygosity were calculated with Partek Genomics Suite software (Partek, St. Louis, MO, USA). This analysis revealed a shared homozygous region of 4.68 Mb on chromosome 4 in all three affected siblings (see Table S1 available online) containing 15 genes. Subsequently, exome sequencing was performed in two out of three affected siblings (A-II:1 and A-II:2). Exome sequencing was performed on a SOLiD4 sequencing platform (Life Technologies, Carlsbad, CA, USA), and the exomes were enriched according to the manufacturer’s protocol by using Agilent’s SureSelect Human All Exon v.2 Kit (50 Mb), containing the exonic sequences of approximately 21,000 genes (Agilent Technologies, Inc., Santa Clara, CA, USA). LifeScope software v2.1 (Life Technologies, Carlsbad, CA, USA) was used to map color space reads along the hg19 reference genome assembly. The DiBayes algorithm, with high-stringency calling, was used for single-nucleotide variant calling. The small Indel Tool was used to detect small insertions and deletions. For the analyzed individuals, a total of 83,287,089 and 76,841,552 reads were uniquely mapped to the gene-coding regions, respectively, with a median coverage of 67.3× in person A-II:1 and 61.9× in person A-II:2. The analysis uncovered 39,561 variants for individual A-II:1 and 39,029 variants for individual A-II:2. Ninety-six nonsynonymous exonic and canonical splice-site variants, occurring with a frequency of less than 1% in 1,154 unrelated individuals (in-house exome database), were shared by both individuals (data not shown). No pathogenic variants in the genes known to be mutated in arCRD (ABCA4, ADAM9, C8orf37, CERKL, EYS, RPGRIP1, and TULP1) were observed among the shared variants. Under the hypothesis of autosomal recessive inheritance, we did not identify two heterozygous variants in a single gene (>20%–<80% variation) in both affected individuals and only a single homozygous (>80% variation) variant, p.Glu189^∗(c.565C>T [NM_001017979.2]), in RAB28 (MIM 612994). This variant is located in the largest shared homozygous region in family A (Figure 1A; Table S1).

Sanger sequencing confirmed the homozygous presence of c.565C>T in the three affected siblings (Figures 1C and 1D). The mutation was not identified in 176 ethnically matched controls or in the Exome Variant Server (EVS) database (release ESP6500).

Primers to amplify all coding exons and their exon-intron boundaries were designed by using Primer 3 software (Table S2). Sanger sequencing of all coding exons of RAB28 in 468 unrelated CRD and 149 unrelated individuals with cone dystrophy did not identify additional likely pathogenic mutations. We assessed SNP data of >400 unrelated individuals with arCRD, Leber congenital amaurosis (LCA), and retinitis pigmentosa (RP) through the European Retinal Disease Consortium^6,17 and identified seven families with conspicuously large homozygous regions spanning RAB28. Sanger sequencing of RAB28 in these families revealed a homozygous nonsense mutation (c.409C>T [p.Arg137^∗]) in a consanguineous family (family B) of Moroccan Jewish ancestry. The two affected siblings in family B (II:1 and II:2; Figure 1) shared four homozygous genomic regions (over 10 Mb) by whole-genome SNP analysis (Table S1). The largest region was on chromosome 7 and the second largest region was on chromosome 4, the latter including 315 genes among which OPN1SW (MIM 613522), and RAB28. Mutation analysis of OPN1SW did not yield causative variants. Segregation analysis confirmed the presence of the homozygous nonsense mutation in RAB28 in both siblings with CRD (Figures 1C and 1D). This mutation was not identified in 118 ethnically matched controls or in the EVS database.

The clinical features of the affected individuals of both families are described in Table 1. Members of family A were diagnosed with CRD in the second decade of life, with rapidly deteriorating visual acuities and high myopia. At their last visit, fundoscopy showed hyperpigmention of the fovea and autofluorescence revealed a slight hyperfluorescent fovea (Figures 2A and 2B). Optical coherence tomography (OCT) displayed altered photoreceptors in the fovea, whereas the peripheral photoreceptors are intact (Figure 2C). Color-vision tests revealed defects in all axes, and visual field testing showed a central scotoma. On ERG photopic responses were nondetectable and scotopic responses reduced in all three siblings. The clinical presentation of both affected individuals of family B was comparable to that of family A. Individual B-II:1 presented with low vision since early childhood, with progressive deterioration of the visual acuity and high myopia. The retinal pigment epithelium showed foveal atrophy, hypofluorescence was observed in the fovea, and a hyperfluorescent ring was noted around the fovea (Figures 2D and 2E, respectively). OCT imaging confirmed absence of photoreceptors in the central fovea, whereas the photoreceptor layer further out appears largely intact (Figure 2F). Color-vision defects were noted in both siblings. Photopic ERG responses were nondetectable, and scotopic responses were moderately reduced.

RAB28 encodes a member of the Rab (“Ras-related in brain”)¹⁸ subfamily of the RAS-related small GTPases. Rab GTPases are key regulators in trafficking of proteins and function as molecular switches to regulate fusion steps in vesicle transport, vesicle budding, motility, tethering,¹⁹ and membrane fission.²⁰ Proper membrane association and subcellular localization of Rab proteins requires posttranslational prenylation of cysteine motif(s) at the carboxyl terminus, which for the majority of Rabs involves the attachment of a geranylgeranyl (20 carbons) anchor by geranylgeranyltransferase II (GGT2 or RabGGT). RAB28 is a rather distant relative of the at least 60 different Rabs,²¹ standing out because of its limited similarity (32% amino acid identity) to other Rab GTPases and its specific amino acid motifs.^21–23 RAB28 expresses three mRNA variants encoding different RAB28 isoforms with alternative C-termini (see Figure 1B).²² Another unique feature of RAB28 is that isoforms 1 and 2 are predicted to be farnesylated at their CaaX-motifs,^22–25 which was experimentally proven for isoform 1 of RAB28.²⁵ The third isoform does not contain a CaaX-motif and therefore cannot be prenylated.

Wide expression is found for variant 1 in contrast to variant 2, which has been reported to be primarily expressed in testis.²² Expression analysis in RNA samples from various human tissues showed highest expression of variant 1 in lung, bone marrow, retinal pigment epithelium (RPE), and kidney, wide and abundant expression of variant 2, and highest expression of variant 3 in heart, lung, bone marrow, retina, brain and RPE (Figure S1). Staining of rat retina showed localization of RAB28 to the basal body and the ciliary rootlet of the photoreceptors (Figure 3; Figure S3).

Both nonsense mutations identified in the CRD families in this report are found in the shared mRNA segment, and are therefore predicted to truncate all three RAB28 isoforms. To determine whether the RAB28 mRNA is subjected to nonsense-mediated decay (NMD), RT-PCR analysis was performed in RNA samples isolated from lymphocytes of affected individuals of families A and B by using a primer set amplifying exons 2 and 3, a region that is shared by all three RAB28 isoforms, and three different primer sets to evaluate the expression of the three isoforms individually. RAB28 transcript was detected in affected members of both families, excluding complete loss of RAB28 transcript by NMD (Figure S2).

The exact role of RAB28 in cone and rod photoreceptors remains to be determined. The localization of RAB28 to the basal body and ciliary rootlet suggests a role in ciliary transport. Interestingly, RAB8 and RAB11, which have so far not been shown to be associated with disease, coordinate primary ciliogenesis²⁸ and together with RAB3A and RAB6A are involved in rhodopsin transport from the photoreceptor inner to outer segments through the connecting cilium.^29–32 RAB28 may have adopted a similar function with specific importance for the highly active vesicle-based transport of photoreceptor proteins through the connecting cilium. Identification of RAB28 mutations emphasizes the role of disrupted ciliary processes in the pathogenesis of CRD. In addition to RAB28, three proteins involved in CRD (C8orf37, RPGRIP1, and TULP1) have so far been shown to localize to the connecting cilia of photoreceptor cells, and approximately one third of the gene defects underlying retinal degeneration affect the connecting cilium structure and/or function in photoreceptors.³³ The observation that nonsense mutations in RAB28 cause a phenotype only in the eye may point to functional redundancy in other tissues.

In conclusion, a combined approach of homozygosity mapping and exome sequencing identified nonsense mutations in RAB28 as a rare cause of arCRD. This study reveals a crucial role for RAB28 in photoreceptor function and suggests that mutations in other Rab proteins may also be associated with retinal dystrophies. The functional implications of RAB28 mutations remain unclear and warrant further investigation.

Supplemental Data

Web Resources

The URLs for data presented herein are as follows:

• Exome Variant Server (EVS) database, release ESP6500, http://evs.gs.washington.edu/EVS/

• Online Mendelian Inheritance in Man (OMIM), http://www.omim.org/

• Primer3, http://frodo.wi.mit.edu/primer3/