retinitis pigmentosa ( rp ) is a heterogeneous group of inherited retinal disorders characterized by night blindness , constricted visual fields , abnormal color vision , and retinal degeneration .
the prevalence of rp is approximately 1 per 4000 persons , with more than 1 million affected individuals existing worldwide .
rp patients show various inheritance patterns including autosomal recessive , autosomal dominant , x - linked , mitochondrial , and digenic   inheritance .
autosomal dominant rp ( adrp ) makes up 30 to 40% of the overall rp cases , while mutations in the rhodopsin ( rho ) gene are responsible for about 25% of adrp cases found in caucasians .
the rho gene has been mapped to the long arm of chromosome 3 ( 3p21 - 24 ) and encodes 348 amino acids . in 1990 ,
the rho gene was first described in the literature as being the causative gene for adrp [ 5 , 6 ] .
while rhodopsin is a typical seven transmembrane g - protein - coupled receptor , a photon and not a molecular ligand is responsible for initiating the rhodopsin phototransduction cascade . when rhodopsin absorbs the photon , retinal chromophore ( 11-cis - retinal ) changes to all - trans - retinal .
the conformational changes that occur in rhodopsin result in the hyperpolarization of the rod cells , which play an important role in vision .
dominant ( or heterozygous ) rho mutations have been reported to show two different rp phenotypes , classic rp and sector rp [ 812 ] .
classic rp is a typical form of rp that is characterized by early - onset and diffuse / generalized retinal dysfunction , whereas sector rp is characterized by adult - onset and regionalized / sectorial retinal dysfunction [ 1315 ] .
sector rp , as originally described by bietti in 1937 , is characterized by retinal degeneration that is limited to one or two quadrants of the fundus and slowly progression compared with classic rp [ 1315 ] .
for example , there is a much lower frequency of rho mutations in japanese , chinese , and korean populations compared with european populations [ 1719 ] .
found rho mutations in 1/13 ( 7.7% ) adrp japanese patients , whereas a separate study found that 43/150 ( 29% ) adrp patients in north america had the rho mutations .
thus , rho mutations have not been considered a major cause of adrp in japanese patients . to date
, only a small number of rho mutations have been reported in the japanese population [ 20 , 2225 ] . in our current study
, we used a whole - exome sequencing technique and identified two rho mutations in two japanese families with adrp , one ( p.w126l ) of which was novel .
we additionally examined the impact of the p.w126l mutation on rhodopsin conformation by investigating the molecular modeling .
the protocol of this study was approved by the institutional research board of the jikei university school of medicine and national hospital organization tokyo medical center .
the protocol adhered to the tenets of the declaration of helsinki , and informed consent was obtained from all participants .
rp diagnosis was based on the visual field , fundus examination , and electroretinogram ( erg ) findings .
detailed ophthalmic examinations were conducted in the two families that exhibited the rho mutations ( family 1 : ju#0678 - 062jikei and family 2 : ju#0575 - 037jikei ) ( figures 1(a ) and 1(b ) ) .
these evaluations included decimal best - corrected visual acuity ( bcva ) , slit - lamp , and fundus examinations , optical coherence tomography ( oct ) ( cirrus hd - oct ; carl zeiss meditec ag , dublin , ca ) , and fundus autofluorescence imaging ( spectralis hra ; heidelberg engineering , heidelberg , germany ) .
visual fields were assessed with kinetic goldmann perimetry ( gp ; haag streit , bern , switzerland ) .
full - field erg was performed according to the protocols of the international society for clinical electrophysiology of vision .
details of the methods and normal data have been reported previously . after obtaining venous blood samples from ten adrp patients ,
whole - exome sequencing was performed in all ten adrp patients using a previously described method .
the obtained sequence data in the patients were compared with reference human genome sequences ( 1000 genomes phase 2 reference , hs37d5 ) .
subsequently , we then focused on only the variants that could change the amino acid sequence , such as the nonsynonymous variants , splice acceptor , and donor site variants , and the short insertions and deletions . in the next step
, we filtered the remaining variants using the criteria that the frequency of the variant had to be less than 1% in the databases of the 1000 genomes project ( http://www.1000genomes.org ) and the human genetic variation browser ( http://www.genome.med.kyoto-u.ac.jp/snpdb/index.html ) . in the final step , we screened variants residing within the 212 retinal disease - associated genes listed in the retnet database that was last updated on march 10 , 2014 ( https://sph.uth.edu/retnet/ ) .
sanger sequencing for rho mutations was conducted in two of the japanese families , which included probands and other family members ( figures 1(a ) and 1(b ) ) .
we used two primer pairs : a forward primer ( rho-2f ) , 5-ctcctcaaatccctctcccactcct-3 , and a reverse primer ( rho-2r ) , 5-tcttctgccctacacccctaccctg-3 for exon 2 , and a forward primer ( rho-5f ) , 5-cgaacctcactaacgtgccag-3 , and a reverse primer ( rho-5r ) , 5-ggtcttggtggatgtcccttc-3 for exon 5 .
the models for molecular dynamics ( md ) simulation were generated using two bovine rhodopsin / opsin crystal structures as the templates : the dark - adapted rhodopsin ( protein data bank i d : 1u19 , chain a ) and the ligand - free form opsin ( protein data bank i d : 3cap , chain a ) . to examine the impact of the p.w126l mutation on protein conformation ,
amino acids in these templates were replaced with the corresponding amino acids from the human sequence , thereby resulting in the wild - type ( wt ) models .
the p.w126l models were generated from the wt models by changing the tryptophan at position 126 to leucine .
all amino acid replacements were performed by using the simple mutate function of the coot software .
the membrane environment was an artificially generated palmitoyl - oleoyl - phosphatidyl choline bilayer of approximately 120 molecules with a dimension of 80  80   .
all of the models we developed were superposed onto each other so that each model was embedded in the artificial membrane at almost identical orientations .
the protein - membrane system was then solvated with water containing 150  mm nacl .
md simulations were run by the not ( just ) another molecular dynamics program   through the visual molecular dynamics interface using the following conditions : 1 fs per step , 100,000 steps ( 100 ps ) for minimization , and 1,000,000 steps ( 1  ns ) , periodic boundary conditions and particle mesh ewald method , cut - off at 10   , and switching at 9   .
the calculations were carried out under a constant pressure and temperature ensemble at 310  k and 1 bar .
a figure was prepared using the discovery studio visualizer software ( accelrys inc . , san diego , ca ) .
patient ii-2 ( a proband ) was a 58-year - old man , who was referred to our hospital because of suspicion of rp .
bcva at his first visit to our hospital was 1.5 ( with + 1.50 diopter ( dpt ) , cylinder ( cyl ) 1.25 dpt axial ( ax ) 20 ) in his right eye and 0.7 ( with + 1.50 dpt .
no abnormalities were found except for slight senile cataracts in the anterior segments and media of both eyes .
fundus examination revealed retinal degeneration around the inferior vascular arcade in his right ( figure 2(a ) ) and left eyes .
the oct images showed marked thinning of the outer nuclear layer ( onl ) and disruption of the inner segment / outer segment ( is / os ) line , except for the foveal region of his right ( figure 2(a ) ) and left eyes .
gp at a previous hospital showed depression of visual fields and an arcuate scotoma at the superior visual field in his right ( figure 2(a ) ) and left eyes .
full - field erg showed that there were decreased amplitudes in the rod , standard combined , cone , and 30-hz flicker responses ( figure 3(a ) ) .
the clinical findings of patient iii-1 were essentially similar to those found for patient ii-2 .
35 ) in her left eye . there were no abnormalities found in the anterior segments and media of either eye .
fundus examination revealed retinal degeneration in the nasal area of her right ( figure 2(b ) ) and left eyes .
the oct images showed marked thinning of the onl and disruption of the is / os line except for the foveal region of her right ( figure 2(b ) ) and left eyes .
gp showed several isolated scotomas in her right ( figure 2(b ) ) and left eyes .
full - field erg showed that there were decreased amplitudes in the rod , standard combined , cone , and 30-hz flicker responses ( figure 3(b ) ) .
patient ii-1 ( a proband ) was a 35-year - old woman referred to our hospital for assessment of a causative gene for her rp .
bcva was 0.7 ( with no correction ) in her right eye and 0.6 ( with cyl .
30 ) in her left eye . there were no abnormalities found in the anterior segments and media of either eye .
fundus examination revealed diffuse retinal degeneration and intraretinal pigment deposits with a bone - spicule configuration around the vascular arcade to the periphery of her right ( figure 2(c ) ) and left eyes .
the oct images showed marked thinning of the onl and entire disruption of the is / os line in her right ( figure 2(c ) ) and left eyes .
gp showed a ring - like defect in her right ( figure 2(c ) ) and left eyes .
full - field erg showed that there were no responses in the rod , standard combined , and 30-hz flicker erg ( figure 3(c ) ) .
patient iii-1 was a 14-year - old boy , who was first found to have night blindness at 6 years of age . at the age of 11
, a dark - adapted single flash erg performed at another hospital showed that there were reduced responses in both eyes ( data not shown ) .
180 ) in his right eye and 1.0 ( with + 7.00 dpt . , cyl . 1.50
fundus examination revealed retinal degeneration of the inferotemporal areas in his right ( figure 2(d ) ) and left eyes .
the oct images showed that there was marked thinning of the onl and disruption of the is / os line , except for the foveal region of his right ( figure 2(d ) ) and left eyes .
gp showed constriction of visual fields with small scotomas in his right ( figure 2(d ) ) and left eyes .
a fundus autofluorescence image revealed a perifoveal hyperautofluorescent ring in his right ( figure 2(e ) ) and left eyes .
the obtained sequence data were analyzed in accordance with the filtering steps discussed in the material and methods section .  
table 1 summarizes the remaining rare variants that were examined in families 1 and 2 .
the analysis focused on the 212 retinal disease - causing genes that were listed in the retnet database .
the two rho mutations revealed in the data , c.377g > t , p.w126l in exon 2 and c.1036g > c , p.a346p in exon 5 , were found in the adrp patients of families 1 and 2 , respectively . with the exception of these rho mutations
, there were no other mutations found in the obtained sequence data for both families 1 and 2 that fulfilled the rp phenotype and autosomal dominant inheritance pattern conditions .
the p.w126l mutation has not been previously reported in the literature and is not found in the dbsnp ( http://www.ncbi.nlm.nih.gov/snp/ ) , 1000 genomes database , or the hgmd ( http://www.hgmd.org ) .
on the other hand , the p.a346p mutation was previously reported to be the cause of adrp in one spanish family .
we used in silico bioinformatics tools to investigate the impact of the p.w126l mutation on the rho function .
results of the polyphen-2 program generated a score of 0.999 ( probably damaging ) , which was close to the maximum value of 1.00 , while the sift program generated a score of 0 ( damaging ) .
for the cosegregation analysis , we investigated whether six of the family 1 members and four of the family 2 members had either the p.w126l or the p.a346p mutation .
our results revealed there was complete cosegregation of each mutation with the rp phenotype in each family ( figures 1(a ) and 1(b ) ) . by using energy minimization and md simulations
, we investigated the possible effects of the p.w126l mutation on the structure of the human opsin moiety that was represented by either the dark - adapted like ( high affinity for 11-cis - retinal ) or the light - adapted like ( high affinity for all - trans - retinal ) crystal structures . although the simulation time was limited , all four models examined approached similar levels of equilibria . because the mutation site w126 is located in the third transmembrane helix ( tm3 ) , we analyzed differences in the tm3 backbone and in the nearby helices . in the dark - adapted template , the extracellular and central regions of tm3 did not appear to move significantly during the simulation regardless of the type of side chain ( w or l ) at position 126 ( figures 4(a ) and 4(b ) ) .
this result corroborates the previous observation that the p.w126l mutation affected the 11-cis - retinal binding only marginally in cos-1 cells .
interestingly , in the light - adapted like template , the tm3 helix within the heptahelical bundle moved differently in the wt and the p.w126l mutant models during the 1  ns simulation ( figures 4(c ) and 4(d ) ) . while the wt and the p.w126l mutant models exhibited only marginal changes in the tm3 during the first 300  ps of simulation ( data not shown ) , this helix progressively tilted only in the p.w126l mutant during the subsequent 1  ns of simulation ( figure 4(d ) ) .
persistence of this tilting during longer periods may lead to a decrease in the distance between the cytoplasmic ends of tm3 and tm6 and may affect the signal - transduction properties of the protein .
the residue l125 , which is next to w126 in bovine rhodopsin , is in contact with a highly conserved phenylalanine in the middle of the tm6 at residue 261 .
these side - chain interactions may therefore be critical for the ligand - induced activation of the rhodopsin - like g - protein - coupled receptors , similar to that which has been proposed for the 2-adrenergic receptor .
in this study , we identified two rho mutations ( p.w126l and p.a346p ) as disease causes by using whole - exome sequencing in two japanese families with adrp .
we additionally used molecular modeling to analyze the impact of the novel mutation ( p.w126l ) on the protein structure and function and then evaluated the genotype - phenotype correlations among japanese rp patients with heterozygous rho mutations . for the cosegregation analysis ,
further validation by sanger sequencing in other family members demonstrated there was complete cosegregation of each mutation with the rp phenotype ( figures 1(a ) and 1(b ) ) .
for the novel p.w126l mutation , the tryptophan residue at the position 126 is located in the tm3 and is highly conserved among orthologs in vertebrates ( figure 1(c ) ) . a biochemical experiment using reconstituted bovine rhodopsin
has shown that an analog of 11-cis - retinal is cross - linked to the w126 residue .
our in silico study , which used the polyphen-2 and sift programs , predicted that the p.w126l mutation would cause severe damage to the rhodopsin .
in addition , the results of our protein modeling and simulations ( figure 4 ) suggest that the p.w126l mutation will likely affect the side - chain interaction between tm3 and tm6 in the light - adapted form but will not affect the interaction in the dark - adapted form .
these molecular modeling and simulation findings suggest that the p.w126l mutation may impair rhodopsin function by affecting its conformational transition in the light - adapted form .
this interpretation is in line with previous studies that have shown that p.w126l of bovine rhodopsin is less potent during g - protein activation .
thus , these results predict that the p.w126l mutation can affect the rho function , especially in the light - adapted form , thereby leading to the phenotype of rp . on the other hand ,
p.a346p has previously been reported to be an adrp - causing mutation in one spanish family .
both our cosegregation data for each family and the molecular modeling confirm that p.w126l is a disease - causing mutation .
with regard to the phenotypes of our patients , the p.w126l ( family 1 ) and p.a346p ( family 2 ) mutations are likely to be associated with sector rp and classic rp , respectively .
however , it should be noted that patient iii-1 ( family 2 ) was not diagnosed with either classic or sector rp , as patient iii-1 exhibited an early stage of rp .
the phenotypes of patient ii-1 ( family 2 ) were similar to those previously reported for a patient of european descent who carried the p.a346p mutation and exhibited classic rp .
these findings suggest that the p.a346p mutation might be associated with the phenotype of classic rp . when trying to diagnose classic or sector rp , it is highly important that one understands the severity and prognosis of rho - associated adrp .
as compared to classic rp , sector rp is considered to be a less severe disease with subnormal erg and visual field defects that correspond to the affected sectors [ 13 , 14 ] .
in fact , it has been reported that sector rp is caused by a number of rho mutations , including p.t4k , p.n15s [ 3941 ] , p.t17 m [ 33 , 42 ] , p.p23h , p.t58r , p.n78i , and g106r [ 11 , 25 , 44 ] . to the best of our knowledge
, there have been nine rho mutations reported in the japanese adrp population [ 20 , 2225 , 32 , 33 , 41 ] , with detailed phenotypes described in five ( p.n15s , p.t17 m , p.g106r , p.e181k , and p.p347l ) out of the nine mutations [ 20 , 2325 , 32 , 33 , 41 ] . among these five rho mutations , one ( p.p347l ) exhibited classic rp while the other four ( p.n15s , p.t17 m , p.g106r , and p.e181k ) showed sector rp .  
table 2 summarizes the clinical features that were described for the genotype - phenotype correlations of the seven japanese adrp families ( including our families ) with the seven rho mutations ( p.n15s , p.t17 m , p.g106r , p.w126l , p.e181k , p.a346p , and p.p347l ) .
interestingly , patients with five other mutations ( p.n15s [ 3941 ] , p.t17 m [ 33 , 42 ] , p.g106r [ 11 , 25 , 44 ] , p.a346p , and p.p347l [ 5 , 32 , 45 ] ) were found to have phenotypes that were similar to japanese and other ethnic groups , although the clinical phenotypes for two other mutations ( p.w126l and p.e181k [ 20 , 21 , 46 ] ) could not be sufficiently evaluated [ 5 , 11 , 20 , 21 , 25 , 3234 , 3942 , 4446 ] .
these findings suggest that the location of each missense mutation is important for the purpose of predicting a diagnosis of either classic or sector rp and that there is a similarity of phenotypes between japanese and other ethnic group rp patients who have identical rho mutations .
report the phenotype - genotype correlations between adrp patients with rho mutations , revealing that patients with mutations altering the intradiscal domain near the n - terminal region ( or a low - numbered codon ) tended to have better visual function than patients with mutations altering the cytoplasmic domain near the c - terminal region ( or a high numbered codon ) ; patients with a mutation altering the transmembrane domain or a mid - numbered codon had intermediate function .
thus , identification of rho mutations appears to be useful for predicting the severity of the rp phenotypes and providing precise genetic counseling . in conclusion
, we identified two rho mutations ( p.w126l and p.a346p ) in two japanese families with adrp .
the genotype - phenotype correlations indicated that the location of the rho mutations is likely to determine the phenotype of either classic or sector rp .
identification of rho mutations is a very useful tool for predicting the disease severity and providing precise genetic counseling .
 
 purpose . to investigate genetic and clinical features of patients with rhodopsin ( rho ) mutations in two japanese families with autosomal dominant retinitis pigmentosa ( adrp ) 
 . methods . 
 whole - exome sequence analysis was performed in ten adrp families . 
 identified rho mutations for the cosegregation analysis were confirmed by sanger sequencing . 
 ophthalmic examinations were performed to evaluate the rp phenotypes . 
 the impact of the rho mutation on the rhodopsin conformation was examined by molecular modeling analysis . results . in two adrp families , we identified two rho mutations ( c.377g > t ( p.w126l ) and c.1036g > c ( p.a346p ) ) , one of which was novel . 
 complete cosegregation was confirmed for each mutation exhibiting the rp phenotype in both families . 
 molecular modeling predicted that the novel mutation ( p.w126l ) might impair rhodopsin function by affecting its conformational transition in the light - adapted form . 
 clinical phenotypes showed that patients with p.w126l exhibited sector rp , whereas patients with p.a346p exhibited classic rp . conclusions . 
 our findings demonstrated that the novel mutation ( p.w126l ) may be associated with the phenotype of sector rp . 
 identification of rho mutations is a very useful tool for predicting disease severity and providing precise genetic counseling . 