A novel RLBP1 gene geographical area-related mutation present in a young patient with retinitis punctata albescens
Scimone et al. Human Genomics
A novel RLBP1 gene geographical area- related mutation present in a young patient with retinitis punctata albescens
Concetta Scimone 0 2
Luigi Donato 0 2
Teresa Esposito 1
Carmela Rinaldi 0
Rosalia D'Angelo 0
Antonina Sidoti 0 2
0 Department of Biomedical and Dental Sciences and Morphofunctional Imaging, Division of Molecular Genetics and Preventive Medicine, University of Messina , via C. Valeria 1, I-98125 Messina , Italy
1 Department of Experimental Medicine, Division of Human Physiology and Integrate Biological Functions “F. Bottazzi”, University of Campania Luigi Vanvitelli, ex II University of Naples , via Santa Maria di Costantinipoli 16, I-80138 Naples , Italy
2 Department of Cutting-Edge Medicine and Therapies, Biomolecular Strategies and Neuroscience, Section of Neuroscience-applied Molecular Genetics and Predictive Medicine, I. E. ME. S. T , via Michele Miraglia 20, I-90139 Palermo , Italy
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.
Retinitis punctata albescens; RLBP1; Frameshift mutation; Population study; Geographical-area related mutation; RP mutation spectrum
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 [
these, the retinaldehyde-binding protein 1 (RLBP1)
mutations are cause of retinitis punctata albescens
(RPA, OMIM#136880) [
]. 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
]. 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 [
]. However, RPA cases
associated with Rhodopsin (RHO), Retinol Dehydrogenase 5
(RDH5), Peripherin 2 (PRPH2), and Lecithin Retinol
Acyltransferase (LRAT) genes were also reported [
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 [
]. A founder effect
was proven for mutation p.R234W, causing Bothnia
], as well as for splice-junctions mutations
detected in NFRCD cases [
]. 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
]. This process is common in both cones
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
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.
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.
Effects of the novel mutation on CRALBP tertiary
structure were predicted by RaptorX (http://raptorx.uchica
go.edu/) prediction tool.
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
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 χ2test with 2 × 3 contingency
tables and 1 degree of freedom. Analysis was performed
by the IBM SPSS statistical analysis software.
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
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
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
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.
About other family’s members, the novel mutation was
observed in both parents (Fig. 1, III:5, III:6), in heterozygous
condition (Fig. 3b) and was absent in the sister (Fig. 1, IV:8;
Fig. 3c). Moreover, mutated allele was found both in
paternal and in maternal relatives (Fig. 1, II:2, II:3, II:7, III:8).
As previously described, RLBP1 mutations are very
uncommon and founder mutations were reported. Frequency of
c.398delC allele was assessed in both Fiumedinisi and
Sicilian population. In the first group, the mutated allele
was detected in 0.01%, while none of screened samples of
general Sicilian population showed the variant. Table 1
shows the distribution of allele frequencies in Fiumedinisi
population; χ2 test results in a p value = 0.9306 showing the
condition of HWE (HWE not consistent for p < 0.05).
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
A total of 300 samples were genotyped. Only 0.01% of screened population carries the mutated allele. Chi-square test exhibits a p value = 0.9306 suggesting the
presence of the equilibrium’s condition between wild-type and mutated allele in Fiumedinisi population
stop codon falls within exon 8. CRALBP protein contains
one CRAL-TRIO lipid-binding domain, of 162 aminoacids
(136-297). 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 [
]. 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 km2 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.
Still today, it results in not being easy to get to. Two big
disasters hit the city: an epidemic plague in 1743 and a big
flood in 1855. A period of decline, then, started due to
high immigration’s phenomenon. To date, Fiumedinisi’s
population counts about 1400 inhabitants, most of which
are over the age of 50 years. For our aim, we screened
about 25% of population and c.398delC was found in 0.01%
and it is in HWE. No other RPA cases were detected in
the town. Although there is a high rate of consanguinity
in Fiumedinisi’s population, its low frequency may be
attributed to several dominations, repeated drastic
reductions in population, the probable selective disadvantage,
and a recent founder effect. This last hypothesis can be
demonstrated by several statistic approaches. Among these,
the method proposed by Slatkin and Rannala [
based on allele frequency and coalescent process  is
what we think to apply to estimate the age of c.398delC
mutation, as next development of this paper.
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.
CRALBP: Cellular retinaldehyde-binding protein; FA: Fundus albipunctatus;
HWE: Hardy-Weinberg Equilibrium; NFRCD: Newfoundland rod-cone
dystrophy; OCT: Optical coherent tomography; RLBP1: Retinaldehyde binding
protein 1; RP: Retinitis pigmentosa; RPA: Retinitis punctata albescens;
RPE: Retinal pigment epithelium
We thank Fiumedinisi’s population for disposability, particularly Dr.Bertani
Gilda for her precious collaboration in controls collection.
Authors received no financial support to perform this study.
Availability of data and materials
All collected data are available contacting corresponding author.
CS wrote the manuscript; LD and TE collected samples and performed
molecular analysis; CR supervised the study; RD’A designed the study;
AS coordinated the research team. All authors read and approved the
Ethics approval and consent to participate
The study involves human participants. Informed consent was fully obtained for
all participants to the study. For underage subjects, consent was obtained from
the parents. The study was conducted in accordance with Declaration of
Helsinki and it was approved on 04/04/2017 by our local Ethics Committee
“Azienda Policlinico Univaersitario G. Martino,” protocol number 2317bis.
The paper, subject of this submission, has not been published or submitted
in total or in part in any language as contribution either to a journal, as book
chapter, or as abstract. All authors read the final version of the manuscript
and they agree for publication.
Consent for publication
Subjects involved in this study were fully informed about its aim. They agree
with the use of clinical information and consent their publication, in
The authors declare that they have no competing interests.
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