A novel RLBP1 gene geographical area-related mutation present in a young patient with retinitis punctata albescens

Background Autosomal recessive forms of retinitis punctata albescens (RPA) have been described. RPA is characterized by progressive retinal degeneration due to alteration in visual cycle and consequent deposit of photopigments in retinal pigment epithelium. Five loci have been linked to RPA onset. Among these, the retinaldehyde-binding protein 1 gene, RLBP1, is the most frequently involved and several founder mutations were reported. We report results of a genetic molecular investigation performed on a large Sicilian family in which appears a young woman with RPA. Results The proband is in homozygous condition for a novel RLBP1 single-pair deletion, and her healthy parents, both heterozygous, are not consanguineous. Thenovelc.398delC (p.P133Qfs*258) involves the exon 6 and leads to a premature stop codon, resulting in a truncated protein entirely missing of CRAL-TRIO lipid-binding domain. Pedigree analysis showed other non-consanguineous relatives heterozygous for the same mutation in the family. Extension of mutation research in the native town of the proband revealed its presence also in healthy subjects, in a heterozygous condition. Conclusions A novel RLBP1 truncating mutation was detected in a young girl affected by RPA. Although her parents are not consanguineous, the mutation was observed in a homozygous condition. Being them native of the same small Sicilian town of Fiumedinisi, the hypothesis of a geographical area-related mutation was assessed and confirmed.


Background
Retinitis pigmentosa (RP) includes more than 70 different forms of inherited eye disorders characterized by progressive vision loss due to photoreceptors degeneration. This heterogeneity is caused by the high number of genes involved in disease's development. Therefore, each RP form differs from another for causative gene, inheritance pattern, symptomatology, age onset, and clinical features. About genetics, more than 50% of cases are affected by autosomal recessive forms due to mutations in almost 50 loci [1]. Among these, the retinaldehyde-binding protein 1 (RLBP1) mutations are cause of retinitis punctata albescens (RPA, OMIM#136880) [2]. RPA is a progressive retinal degeneration belonging to the group of rod-cone dystrophies and has an early onset characterized by night blindness. At diagnosis, fundus appears with punctate white-yellow deposits. Progressive macular atrophy is the main cause of visual acuity loss, and narrowing of the visual field may occur in late teenage [3]. RPA has an incidence of 1/800000 people worldwide, and mutations at RLBP1 gene were reported only in about 1% of patients affected by autosomal recessive forms [2]. However, RPA cases associated with Rhodopsin (RHO), Retinol Dehydrogenase 5 (RDH5), Peripherin 2 (PRPH2), and Lecithin Retinol Acyltransferase (LRAT) genes were also reported [4][5][6]. About RLBP1 gene, different mutations are linked to a wider spectrum of phenotype as Bothnia retinal dystrophy (BD), Newfoundland rod-cone dystrophy (NFRCD), and fundus albipunctatus (FA). These forms differ for age of onset, progression, and for severity [7]. A founder effect was proven for mutation p.R234W, causing Bothnia phenotype [8], as well as for splice-junctions mutations detected in NFRCD cases [9]. So, different clinical manifestations are tightly linked to the effects of mutations on protein's structure. RLBP1 is expressed in retinal pigment epithelium (RPE) and encodes for the cellular retinaldehyde-binding protein 1, CRALBP, involved in visual cycle. Particularly, CRALBP binds 11cis-retinol which needs to be oxidized in 11-cis-retinal; 11-cis-retinal is carried into photoreceptors where it combines with opsin, resulting in visual pigments formations [10]. This process is common in both cones and rods.
We describe a novel RLBP1 frameshift mutation, detected in a homozygous condition in a young 31-years-old woman, affected by RPA, certainly with no consanguineous parents up to the 4th generation ancestor, and native in Fiumedinisi, a small Sicilian town in province of Messina. Fiumedinisi is entered in a valley of Nebrodi mountains, at a height of about 200 above sea level. Homozygous condition for a novel mutation is a rare event in patients whose parents are not consanguineous; however, in this case, parents are both native of Fiumedinisi. This leads us to hypothesize that detected mutation is a geographical arearelated variant.

Case analysis Case description
Here we present a case of a 31-years-old woman (Fig. 1, IV:7), born in Fiumedinisi, a small Sicilian (Italy) town. Her parents, also native of Fiumedinisi, are not consanguineous. About the proband, at the age of five, myopia was diagnosed; two years later, she began suffering from night vision disturbance. Fundus revealed the typical "salt and pepper" aspect ( Fig. 2a), without macula involvement; additionally, optical coherent tomography (OCT) examination showed the presence of numerous well-demarcated homogenous dome-shaped lesions, originating from the RPE layer (Fig. 2b). One year later, fundus analysis confirmed the progressive kind of phenotype, so RPA was diagnosed. Thereafter, she has report continuous worsening with also reduction of the visual field due to "retinal-tapetum" degeneration ( Fig. 2c). No evidences of eye diseases were reported neither for her parents nor for younger sister.

Molecular analysis
DNA was extracted from peripheral blood. Exons and exon-intron bourdaries of RLBP1, PRPH2, RHO, LRAT, and RDH5 genes were amplified by polymerase chain reaction (PCR). Primers sequences and PCR conditions are available upon request. Direct sequencing was performed using BigDye Terminator v3.1 chemistry and sequencing was ran on a 3500 Genetic Analyzer (Applied Biosystems). Molecular screening of all genes was performed in the proband; then, detected variants were also searched in the other family members.

Population study Samples collection
To test the frequency of novel c.398delC variant, two groups made up by 300 and 600 subjects, respectively, heterogeneous for age and gender, were collected. The first one was recruited in Fiumedinisi and sampling criteria include Fiumedinisi native ancestors by at least Fig. 1 Familial pedigree. The arrow indicates the proband (IV:7). Empty squares and circles symbolize wild-type men and women, respectively. Half fill indicates the heterozygous condition for the c.398delC mutation. Question mark indicates the unknown genotype due to unavailability of the sample. Slash indicates died individuals three generations, absence of consanguinity, and absence of inherited disabling ocular diseases. The second group was collected in the Sicilian area: Ionic and Tyrrhenian municipality in province of Messina, like Santa Teresa Riva, Roccalumera, Pace del Mela, were excluded due to their geographic localization, since destinations of the inhabitants of Fiumedinisi who have left their native town. For each population, the c.398delC allele frequency was calculated as [1 × (h + 2H)]/2 N, where h represents the heterozygous genotype, H thehomozygous genotype, and N the sample size for each population. Deviation from Hardy-Weinberg equilibrium (HWE) was determined using the χ 2 test with 2 × 3 contingency tables and 1 degree of freedom. Analysis was performed by the IBM SPSS statistical analysis software.

Molecular analysis
Buccal swabs were used for collecting saliva samples. DNA was purified by QIAamp® DNA Mini kit (Qiagen). Exon 6 of RLBP1 gene was amplified by PCR for all collected samples, then sequenced on 3500 Genetic Analyzer (Applied Biosystems), using BigDyeTermitor v3.1 chemistry.
All patients and controls involved in this study were fully informed and written consent was obtained. For underage subjects, consent was obtained from the parents. The study protocol followed the guidelines of our local ethics committee, and the investigation was conducted with the ethical requirements defined in the Helsinki Declaration.

Case analysis
Direct sequencing of all known RPA causative genes LRAT, RDH5, and RHO showed no significant variants. Two SNPs rs390659 (c.910C > G, p.Q304E) and rs434102 (c.1013A > G, p.D338G) were detected in PRPH2; however, in ClinVar database, these are reported as benign (http://www.ncbi.nlm.nih.gov/clinvar/variation/138904/, http://www.ncbi.nlm.nih.gov/clinvar/variation/138906/). A novel single base-pair frameshift deletion, the c.398delC (p.P133Qfs*258), was detected in exon 6 of RLBP1 gene in a homozygous condition (Fig. 3a). It is not present in mutations and SNPs databases as HGMD, dbSNPs, ClinVar, ExAC, and Ensembl. The variant leads to substitution of proline 133 in glutamine and results in a frameshift with consequent premature termination at the 258th codon, in exon 8, causing the loss of the last 60 amino acids at the C-terminus of the protein. Figure 4 shows the results of insilico prediction of tertiary structure alteration in mutated protein (Fig. 4b) compared to wild-type one (Fig. 4a). As evident, the c.398delC results in total disruption of functional domain as well as in an altered folding.

Population study
As previously described, RLBP1 mutations are very uncommon and founder mutations were reported. Frequency of  Table 1 shows the distribution of allele frequencies in Fiumedinisi population; χ 2 test results in a p value = 0.9306 showing the condition of HWE (HWE not consistent for p < 0.05).

Discussion
This study reports the results obtained by a molecular analysis performed on a large Sicilian family in which a case of RPA was diagnosed. Molecular screening of all known RPA causative genes showed a novel single basepair frameshift deletion, c.398delC, in exon 6 of RLBP1. It leads to a truncated 258 amino acid protein. Premature  As predicted by in-silico analysis, the c.398delC affects proline 133 and causes total disruption of CRAL-TRIO domain, resulting in a loss of ability to bind its ligands. CRAL-TRIO motifs usually bind small hydrophobic molecules. In CRALBP, aminoacids 136-297 form CRAL-TRIO domain and, 12 of these, form the retinoid binding pocket [11]. Therefore, c.398delC has deleterious effects on protein's functionality.
Presence of variant c.398delC was assessed also in the other family's members; despite, they show no pathological phenotypes. Of these, the healthy father and other relatives showed this variant, in a heterozygous condition. This confirms autosomal recessive model of inheritance of RPA. The proband carries the novel c.398delC mutation in a homozygous condition. We proceeded to investigate frequency of variant in Fiumedinisi's population. Fiumedinisi, a small town in province of Messina, is located on the Ionian side, at the height of 190 above sea level (Fig. 5). It has an area of 36 km 2 and extends along Nisi's valley, at Mt. Belvedere's slopes, behind Valle del Mela. The city was founded in VII century B.C., by a group of Calcidesi Greek colonists, attracted by mineral deposits, who inhabited the high area of Mt. Belvedere. Later, Roman, Arab, Norman, Angevin, and Spanish dominions went on. During these centuries, the town was moved to the foot of mountain (where it remains today) and renamed as FlumenDionisyi.  [12,13] based on allele frequency and coalescent process [14] is what we think to apply to estimate the age of c.398delC mutation, as next development of this paper.

Conclusions
We report a case with RPA caused by the novel mutation, the c.398delC, in RLBP1 gene in homozygous condition necessary for developing the disease. The new mutation enriches the already wide spectrum of RP causative mutations. Hypothesis of a geographic area-related mutation was induced by the detection of this allele in several nonconsanguineous inhabitants and by its absence in Sicilian population. However, due to the succession of more dominations, it is very difficult to establish by which ancestral population it was introduced.