The diagnostic application of targeted re-sequencing in Korean patients with retinitis pigmentosa
Yoon et al. BMC Genomics
The diagnostic application of targeted re-sequencing in Korean patients with retinitis pigmentosa
Chang-Ki Yoon 1 2
Nayoung K. D. Kim 1 5
Je-Gun Joung 5
Joo Young Shin 2
Jung Hyun Park 4
Hye-Hyun Eum 3 5
Hae-ock Lee 5
Woong-Yang Park 0 5
Hyeong Gon Yu 2
0 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine , Seoul , Korea
1 Equal contributors
2 Department of Ophthalmology, Seoul National University College of Medicine , Seoul , Korea
3 Department of Biomedical Sciences, Seoul National University Graduate School , Seoul , Korea
4 Department of Ophthalmology, Seoul Paik Hospital, Inje University , Seoul , Korea
5 Samsung Genome Institute, Samsung Medical Center , Seoul , Korea
Background: Identification of the causative genes of retinitis pigmentosa (RP) is important for the clinical care of patients with RP. However, a comprehensive genetic study has not been performed in Korean RP patients. Moreover, the genetic heterogeneity found in sensorineural genetic disorders makes identification of pathogenic mutations challenging. Therefore, high throughput genetic testing using massively parallel sequencing is needed. Results: Sixty-two Korean patients with nonsyndromic RP (46 patients from 18 families and 16 simplex cases) who consented to molecular genetic testing were recruited in this study and targeted exome sequencing was applied on 53 RP-related genes. Causal variants were characterised by selecting exonic and splicing variants, selecting variants with low allele frequency (below 1 %), and discarding the remaining variants with quality below 20. The variants were additionally confirmed by an inheritance pattern and cosegregation test of the families, and the rest of the variants were prioritised using in-silico prediction tools. Finally, causal variants were detected from 10 of 18 familial cases (55.5 %) and 7 of 16 simplex cases (43.7 %) in total. Novel variants were detected in 13 of 20 (65 %) candidate variants. Compound heterozygous variants were found in four of 7 simplex cases. Conclusion: Panel-based targeted re-sequencing can be used as an effective molecular diagnostic tool for RP.
Retinitis pigmentosa; Targeted re-sequencing; Genetic diagnosis; Familial case; Sporadic case
Retinitis pigmentosa (RP) is a group of hereditary retinal
diseases characterised by progressive loss of rod and
cone photoreceptor cells. It is the most common
hereditary retinal disease with a frequency of approximately 1
in 3000–4000 people, with an estimated total of 1.5
million people affected worldwide [1, 2]. Patients with
RP usually present with a loss of night vision and
complain of progressive constriction of the visual field.
Eventually, central vision is affected and worsens.
Clinical features such as age of onset and rate of
progression vary greatly between individuals.
The identification of causative genetic variants is
expected to be the starting point of RP treatment. Genetic
counselling and gene therapy or other patient-specific
treatment options can be suggested based on genetic
background. In addition to treating the disease,
molecular diagnosis is helpful for informing the differential
diagnosis. Hereditary retinal dystrophies have a slowly
progressive nature and complete clinical features may
not be present at the time of examination. Therefore,
many other hereditary retinal dystrophies may be
confused with RP at certain phases of disease progression.
However, the genetic heterogeneity of RP is a hurdle
for the easy application of molecular diagnosis. At the
time of September 2011, 53 genes and 7 mapped loci are
known to be associated with nonsyndromic RP 
(RetNet). These genes show considerable phenotypic
variability and multiple inherited retinal disease shares
certain causative genes with RP . Because of this
phenotypic heterogeneity and confusion between
genotypic and phenotypic correlations, the clinical features of
individual patients provide few clues to narrow down the
list of candidate genes [2, 5]. Therefore, these genes and
loci should be screened simultaneously.
Massively parallel sequencing, also called next-generation
sequencing (NGS) is a recent technical breakthrough in
genetics [6, 7]. NGS-based high-throughput sequencing
even makes whole-genome sequencing available in a single
facility, which would have required tremendous cost and
time in the past. RP can be a good target for massively
parallel sequencing as an appropriate number of candidate
genes are known for RP. Therefore, this study attempts to
detect candidate variants in RP by applying a targeted
enrichment and re-sequencing strategy.
Quality validation of sequencing
A total of 217.5 kbp of targeting and flanking regions
from 53 RP-related genes and 748 RP-related exons were
captured; 97.1 % of the 2.89 M reads obtained on
average per individual were properly paired and mapped
(Additional file 1: Table S1). The average read depth was
146.2 ± 26.2, and 98.6 % and 96.5 % of the captured
target exons showed more than 1X and 10X coverage,
respectively (Additional file 1: Table S1).
Detection of variants in familial RP
Strong candidate variants of RP were identified in 10
families (Table 1). Among 18 families, 10 had an
autosomal dominant, five had an autosomal recessive and
three had an X-linked inheritance pattern. Briefly, five
known pathogenic variants (c.421-1G > A in PRPF31,
p.P347L in RHO, p.A153V in KLHL7, p.Y178C in RHO,
p.R354X in PRPF31 were detected [8–13]. The known
variant, pC3416G in USH2A, was initially detected, but
Table 1 Strong candidate variants in familiar cases
the other variant, p.G4647R in the XF4 family under the
inheritance pattern of autosomal recessive (compound
heterozygote), was predicted as non-pathogenic.
Therefore, the XF4 family was excluded. The other five
candidate variants in RP1, RP2 and TOPORS have not been
reported in dbSNP and were co-segregated in the family
The 10 variants were comprised of four
nonsynonymous variants, four nonsense mutations, one frameshift
deletion, and one splicing variant. All the nonsense
mutations (p.Y485X in RP1, p.Q766X in RP1, p.R782X in
TOPORS, and p.R354X in PRPF31) were located at the
upstream exons (exon 4 of 4, exon 3 of 4, exon 2 of 3
and exon 10 of 16 respectively), which might lead to
nonsense-mediated decay of mRNA. A frameshift variant
(p. Ser187fs in RP2) was also located at the 5′ upstream
exon, and all the nonsense and frameshift variants were
classified as possible pathogenic variants (category II).
The clinical features were relatively severe in patients
having X-linked variants, F04 (p.C114R in RP2) and F12
(p.Ser187fs in RP2) (Additional file 1: Table S2). The
affected family members experienced early onset of
symptoms and presented with rapid progression. In the
F04 family, the proband (VI-1) was a 6-year-old male
whose visual acuity was 20/200 (OD) and 20/1000
(OS). The visual field was mildly constricted using a
Goldmann perimetry V4 target, and the rod responses
were extinguished and photopic responses were
severely decreased on the electroretinogram. The
photoreceptor inner segment and outer segment junction
was not detectable using OCT (optical coherence
tomography). The patient’s grandfather (II-1) had no
light perception in both eyes. Fundus examination and
OCT scan showed a severely degenerative retina. For
family F12, a possible pathogenic missense variant was
found in the X-linked RP2 gene. Hemizygous male
Xia et al. 
Chr: chromosome; Homo: homozygous; Hetero: heterozygous; Hemi: hemizygous; AD: Autosomal Dominant; X-L X-linked
In-house: Korean normal reference consisting of 192 exomes
patients (II-5, II-16 and III-2) showed poor visual acuity
at less than counting fingers. The retina was totally
degenerated such that the retina and choroid were not
distinguishable on OCT. Scoptopic and photopic
electroretinogram (ERG) were all extinguished. Female
patients of the F12 family (II-8, II-13, III-11 and III-20)
showed variable penetrance. Three of them (II-13, III-11
and III-20) were confirmed to have heterozygous variants.
Two female patients (III-11 and III-20) had mild
peripheral pigmentary deposits and decreased rod and cone
amplitude on electroretinography. Two other female
patients (II-8 and II-13) showed complete expression of
RP. They had visual acuity limited to light perception in
both eyes, typical bony spicule pigment deposits,
attenuated retinal vessels. Scoptopic and photopic ERG
amplitude were also abolished.
F06 (p.Q766X in RP1) showed a milder phenotype.
In family F06, all patients experienced their first
symptoms in their 30s. Two patients older than age 50 (II-3,
75 years old; III-2, 54 years old) had visual loss, while
the other patients (III-6, III-10 and III-13) had normal
vision. Patient III-6 and III-10 showed a normal
macula on OCT and a mildly constricted visual field. The
clinical features of the patients and pedigrees are
summarised in Additional file 1: Table S2 and Figure S1.
Fundus photograph and OCT are presented in Additional
file 1: Figure S2.
Detection of variants in simplex RP
The possible pathogenic variants were also identified in
7 of 16 patients with simplex RP (43.7 %) (Table 2).
These patients denied a familial history of RP or related
hereditary disorders. The inheritance pattern of
candidate genes was based on the sequencing results (Table 2).
Table 2 Strong candidate variants in sporadic cases
Initially four known pathogenic variants, p.H278Y in
PDE6B, p.Y2935X and p.G2186E in EYS, and p.C3416G
in USH2A, were detected. [13–16]. However a possible
second variant in EYS (patient 435) and USH2A (patient
450) was not found, p.Y2935X (EYS) and p.C3416G
(USH2A) were not included in final results. Eight causal
candidate variants were newly identified. Overall, ten
possible pathogenic variants were detected. They were
comprised of six nonsynonymous, three nonsense, and
one frameshift insertion variants. Compound
heterozygous variants (in trans) were detected in five patients
(PDE6B in 436 and 445, EYS in 439 and 440, USH2A in
438). The same frameshift variant of EYS (p.S1653fs)
was identified in two unrelated patients with simplex RP
(439 and 440).
Regarding the clinical feature, patient 430 with the
PRPF31 mutation (p.E104K) showed a mild clinical
phenotype. Although he was 42 years old, his visual
acuity was 20/20 in both eyes, but a paracentral scotoma
was detected in the visual field test. The
electroretinogram showed a small decrease only in rod response.
Most of the photoreceptor inner segment and outer
segment junction (5 mm in the horizontal scan) was
observed intact using an OCT macula scan (Additional
file 1: Table S3). Fundus photograph and OCT are
presented in Additional file 1: Figure S2.
In this study, targeted exome capture and massively
parallel sequencing (MPS) was implemented for genetic
diagnosis of RP. Strong candidate variants were
identified in 10 (55.5 %) of 18 families and 7 (43.7 %) of 16
sporadic patients. The overall detection rate was 50 %.
This method seems to be highly efficient and cost
No Inheritance* Gene
Chr Exon nucleotide
amino acid Mutation Type
EVS In-house Class
PDE6B Compound hetero 4
USH2A Compound hetero 1
Compound hetero 6
PDE6B Compound hetero 4
c.8885 T > G
frameshift insertion Novel
frameshift insertion Novel
Compound hetero 6
*Inheritance is not inferred from pedigrees of the patients. These patients stated that there is no affected individual in their family tree other than indexed
patients. Inheritance pattern described the results suggested by sequencing data
In-house: Korean normal reference consisting of 192 exomes
Chr chromosome; AD Autosomal dominant; AR Autosomal recessive; X-L X-linked
effective in comparison with PCR amplicon sequencing
and other conventional methods such as microarray
analysis. It is certainly much higher than the 7–16 %
detection rate of microarray genotyping [17–21]. A previous
genetic analysis of Korean RP patients using microarrays
revealed a causative mutation in 26 out of 336 patients
(2 %) . The subjects of this microarray study were
different from the current study. Patients from the F09
and F13 families had also been screened for detection of
the RHO mutation by using direct sequencing in a
previous study . The current study analysed 53 target
genes and showed exactly the same results as the direct
sequencing of the RHO gene for these particular
individuals, thereby supporting the accuracy and efficiency of
the current strategy. The current results are also
comparable with other RP genetic analysis using targeted
resequencing, in which the detection rate ranged from
36 to 82 % [23–28]. Unbiased sequencing, including
whole-genome or exome sequencing, can provide
comprehensive genetic data that reveal novel causal genes
of RP such as NEK2, HK1 and MVK. However the
sequencing and data processing burden is still the
bottleneck for widespread use of unbiased sequencing.
Moreover, detection rates using whole exome
sequencing have not proven to be significantly better than
targeted resequencing as previously expected . This
also supports the usefulness of targeted exome
sequencing in RP genetic screening.
Twenty possible causal variants were detected from 10
genes. Of these, 13 variants (65 %) were novel and this
proportion is comparable with recent works reporting
novel allele rates of 62–68 % [25–27, 29]. The
considerable number of novel variants further emphasises the
use of MPS as a reasonable tool for RP molecular
diagnosis. The PRPF31 mutation (17.6 %) is most frequently
found in current study, followed by mutations in EYS,
PDE6B, RHO, RP1, and RP2 (11.8 % respectively). PRPF31
(30 %), RHO (20 %), and RP1 (20 %) were frequently
affected genes in autosomal dominant RP (adRP), whereas
EYS (40 %) and PDE6B (40 %) were frequently affected
genes for autosomal recessive RP (arRP). Despite the
limitation of extending this result to the general distribution
of RP mutations in Koreans, frequently affected genes are
similar to those reported in recent works as well as
previous reports by Hartong et al. [2, 23, 27, 29]. Possible
causal genes responsible for simplex cases had primarily
autosomal recessive inheritance (5 out of 7 cases, 71 %) as
suggested previously [1, 2]. It could not be confirmed
whether causal alleles were de novo because segregation
analysis was not able to be performed in simplex cases.
In X-linked RP, the RPGR gene is thought to be the
most common causal gene, accounting for over 70 % of
cases . Although the RPGR gene was included in the
capture library of the present study, the candidate causal
variant could not be found. ORF15 is a mutation hotspot
of RPGR, where roughly two thirds of disease-causing
mutations are found . This repetitive purine-rich
ORF15 region is rarely covered by next-generation
sequencing. Therefore, the RPGR mutation could have
been missed and, instead, only the RP2 variants detected.
A missense variant (p.C114R) in family F04 and a
truncating variant (p.S187Tfs*31) in family F12 was
identified. The missense variant (p.C114R) was located in the
conserved tubulin binding cofactor C (TBCC) domain
and was evolutionarily conserved. Interestingly, family
F12 showed an autosomal dominant pattern without
male to male transmission. Unlike the expected pattern
of an X-linked disease affecting only males, recent
studies have reported that RP2 and RPGR mutation cause
variable RP phenotypes in heterozygous female
patients.[30–32] Furthermore, it is reported that mutations
in RP2 and RPGR account for 8.5 % of patients with RP
in provisional autosomal dominant families .
PRPF31 variants were identified in two families and
one simplex RP patient. One splicing site variation
(c.421-1G > A in family F07) and truncating variant
(p.R354X in family XF3) were known pathogenic
variants [11, 12]. Haploinsufficiency from a null allele
rather than gain of function is assumed to be the major
mechanism of PRPF31 mutation-derived RP .
However, missense variants also have been suggested as
possibly pathogenic in several reports [34–37]. A
missense variant (p.E104K) was also detected in a simplex
RP patient (430) in the current study. This substitution
is located at the highly conserved NOSIK domain and
this sequence is evolutionarily conserved.
An EYS variant was identified in two simplex patients.
The same frameshift insertion variant (p.S1653Kfs*) was
identified in two unrelated patients. As a second
candidate allele, 439 had a premature truncating variant
(p.E584X) and 440 had a missense variant (p.G2186E).
Nonsense mediated decay from a heterozygous
compound truncating mutation is assumed to be a possible
pathogenic mechanism in 439. The missense variant
(p.G2186E) in patient 440 was located in the highly
conserved Laminin G domain. Interestingly, this mutation
was previously reported in a patient with Korean
ancestry . A frameshift variant (p.S1653Kfs*) and missense
variant (p.G2186E) were recurrent in a relatively small
cohort and it is assumed that these could be founder
variants in Koreans. Iwanami et al. suggested that
patients having a homozygous or compound heterozygous
truncating mutation of EYS show a more severe decline
of visual function than patients having only one allele of
the truncating variant . However, both cases (439
and 440) did not show any difference in progression in
the current study independent from number of
Table 3 53 RP-related genes selected for targeted resequencing
Gene Inheritance RefSeq Cytogenetic Loci Exon Count
ABCA4 AR/AD NM_000350 1p22.1 50
ARL6 AR NM_177976 3q11.2 9
BEST1 AD NM_001139443 11q12.3 9
C2ORF71 AR NM_001029883 2p23.2 2
CA4 AD NM_000717 17q23.1 8
CERKL AR NM_201548 2q31.3 13
CLRN1 AR NM_001195794 3q25.1 4
CNGA1 AR NM_001142564 4p12 10
ATP-binding cassette, sub-family A (ABC1), member 4 (ABCA4)
ADP-ribosylation factor-like 6 (ARL6), transcript variant 2
bestrophin 1 (BEST1), transcript variant 2
chromosome 2 open reading frame 71 (C2orf71)
carbonic anhydrase IV (CA4)
ceramide kinase-like (CERKL), transcript variant 1
clarin 1 (CLRN1), transcript variant 5
cyclic nucleotide-gated channel alpha 1 (CNGA1), transcript
Homo sapiens cyclic nucleotide-gated channel beta 1 (CNGB1),
transcript variant 1
crumbs homolog 1 (Drosophila) (CRB1), transcript variant 1
cone-rod homeobox (CRX)
Homo sapiens dehydrodolichyl diphosphate synthase (DHDDS),
transcript variant 2
eyes shut homolog (Drosophila) (EYS), transcript variant 1
family with sequence similarity 161, member A (FAM161A),
transcript variant 1
fascin homolog 2, actin-bundling protein, retinal
(Strongylocentrotus purpuratus) (FSCN2), transcript variant 2
guanylate cyclase activator 1B (retina) (GUCA1B)
isocitrate dehydrogenase 3 (NAD+) beta (IDH3B), nuclear gene
encoding mitochondrial protein, transcript variant 1
IMP (inosine 5′-monophosphate) dehydrogenase 1 (IMPDH1),
transcript variant 1
interphotoreceptor matrix proteoglycan 2 (IMPG2)
kelch-like family member 7 (KLHL7), transcript variant 1
lecithin retinol acyltransferase (phosphatidylcholine–retinol
c-mer proto-oncogene tyrosine kinase (MERTK)
nuclear receptor subfamily 2, group E, member 3 (NR2E3), transcript
neural retina leucine zipper (NRL)
phosphodiesterase 6A, cGMP-specific, rod, alpha (PDE6A)
phosphodiesterase 6B, cGMP-specific, rod, beta (PDE6B),
transcript variant 1
phosphodiesterase 6G, cGMP-specific, rod, gamma (PDE6G),
transcript variant 1
Homo sapiens progressive rod-cone degeneration (PRCD),
transcript variant 2
prominin 1 (PROM1), transcript variant 1
PRP3 pre-mRNA processing factor 3 homolog (S. cerevisiae) (PRPF3)
PRP31 pre-mRNA processing factor 31 homolog (S. cerevisiae)
PRP8 pre-mRNA processing factor 8 homolog (S. cerevisiae) (PRPF8)
peripherin 2 (retinal degeneration, slow) (PRPH2)
retinal outer segment membrane protein 1
Table 3 53 RP-related genes selected for targeted resequencing (Continued)
A known pathogenic missense variant (p.H278Y) of
PDE6B was detected in two unrelated patients (436 and
445). A truncating variant (p.W11X) was found in
patient 436 as a second variant and a novel missense
variant (p.I256N) was detected in patient 445. This
missense variant is located in the highly conserved GAF
domain. Loss of function of cGMP phosphoodiesterase
activity is a possible mechanism in these two patients.
Just one possible pathogenic allele was found in the
XF4 family and simplex patients 433, 435 and 450. (Data
not shown in Tables 1 and 2) The identified genes in
these patients have an autosomal recessive inheritance
pattern. Known pathogenic variants (p.C3416G of
USH2A in XF4 and 450, p.Y2935X of EYS in 435) were
found in just one allele. As a second pathogenic
candadate, missense variants p.T2465S of EYS and p.V2228E
of USH2A were detected and predicted to be
pathogenic in patient 435 and 450, respectively. (Additional
file 1: Table S4) These were relatively common variants
(more than 1 %) in the genomic data of 1000 Asians.
Considering that the counter allele, p.Y2935X and
p.C3416G, is rare and pathogenic, these candidate
variants may also be pathogenic alleles. Regarding other
cases in the current study (XF4 and 433), a second
pathogenic allele could not be identified. Meaningful
retinol binding protein 3, interstitial (RBP3)
G protein coupled receptor (RGR), transcript variant 1
retinaldehyde-binding protein 1 (RLBP1)
retinitis pigmentosa 1 (autosomal dominant) (RP1)
retinitis pigmentosa 2 (X-linked recessive) (RP2)
retinitis pigmentosa 9 (autosomal dominant) (RP9)
retinal pigment epithelium-specific protein 65 kDa (RPE65)
retinitis pigmentosa GTPase regulator (RPGR), transcript variant C
S-antigen; retina and pineal gland (arrestin) (SAG)
sema domain, immunoglobulin domain (Ig), transmembrane
domain (TM) and short cytoplasmic domain, (semaphorin) 4A
(SEMA4A), transcript variant 2
small nuclear ribonucleoprotein 200 kDa (U5) (SNRNP200)
spermatogenesis-associated 7 (SPATA7), transcript variant 1
topoisomerase I binding, arginine/serine-rich, E3 ubiquitin protein
ligase (TOPORS), transcript variant 1
tetratricopeptide repeat domain 8 (TTC8), transcript variant 1
Tubby-like protein 1 (TULP1)
zinc finger protein 513 (ZNF513), transcript variant 1
second variants may not have been identified because
they were large indels or located in an untranslated
region. Alternatively, detected pathogenic variants might
be accidental carriers and causal variants are located in
other genes. Accidental carrier mutations are
increasingly being reported as the use of multigenic screening
with MPS increases [23, 27]. Although the current
study revealed a possible causative variant in half of
cases, obviously a candidate causal allele could not be
found in the other half of patients. Since the target
library was designed, three more causative genes have
been added to the RP gene list. Unknown causal genes
for RP can still exist and including these targets would
increase the detection yield. However, whole exome
sequencing or the use of an extended target library of
over 100 genes has not revealed a higher detection rate
[25, 29, 38]. Unbiased sequencing not only poses an
enormous data processing burden, but also impedes
deep sequencing. Covering the non-coding exons of
target genes and complete coverage of the target may
be better way to increase diagnostic yield rather than
extending the target library. Eisenberg et al. reported
high detection rates using high coverage, copy number
variance (CNV) analysis and sequencing the 5′ UTR
. Xu et al. reported that complete coverage using
Fig. 1 Schematic workflow of the diagnostic application of targeted
exome sequencing in familial and simplex retinitis pigmentosa. 62
cases consisting of 46 patients in 18 families and 16 cases in simplex
families were recruited, and targeted re-sequencing was performed for
53 RP-related genes. Candidate variants were identified by filtering
based on variant quality > 20 and minor allele frequency < 0.01 from
1000 Genome Project (www.1000genomes.org), Exome Variant Server
(evs.gs.washington.edu) and our in-house DB consisting of 192 Korean
exomes. The variants were finally confirmed by a cosegregation test if
familiar cases, in silico tools and Sanger sequencing
deep sequencing and PCR amplicon for less covered
regions yielded an 82 % detection rate . Although
a ≥ 10X sequencing depth was reached in 96 % of the
sequenced region, 4 % had a less than 10X depth and
1.4 % was not covered (Additional file 1: Table S1).
GC-rich regions or highly repetitive regions are usually
under-captured by MPS. The mutational hotspot,
ORF15 of RPGR, is one of those regions. Complete
Table 4 Classification of candidate variants in this study
Single Nucleotide Variants (SNVs) predicted to cause serious
protein deformity by using in silico analysis
Stopgain, and frameshift mutations
Mutations causing only protein change
coverage assisted by direct sequencing and deep
sequencing is needed to raise the causative variant
Identifying a causal mutation is the starting point of
RP treatment, followed by proper genetic counselling
and prognostic data. For instance, patients can be
informed that they have a certain mutation, which puts
them at risk for rapid progression of RP, and aggressive
therapy can be initiated on the basis of confirmative
genetic data, although there are limited treatment options
available to help RP patients as of yet. Gene therapy, one
of the possible treatment options for RP, is largely
dependent on genetic data, indicating that identifying
the causal mutation will become an increasingly
important step in RP treatment.
Using the targeted re-sequencing of known genes for
RP, pathogenic and possibly pathogenic variants were
identified in more than the half of patients and
families. This strategy will become an increasingly
efficient and cost-effective molecular diagnostic test for
Sixty-two Korean patients with RP were selected for the
RP cohort. This cohort consisted of 46 patients from 18
families and 16 patients with simplex RP. All the
patients had nonsyndromic RP. Comprehensive ocular
examinations, including visual acuity assessment, slit-lamp
examination, fundus examination, electroretinograms,
Goldmann Perimetry (Haag-Streit, Bern, Switzerland)
and optical coherence tomography (Carl Zeiss Meditec
Inc., Dublin, CA, USA) were used for clinical diagnosis.
RP was diagnosed when the patients had the typical
retinal appearance of RP and had attenuated or
abolished rod and cone signal on electroretinography. Bony
spicule-like pigment deposits, retinal vessel attenuation
and optic disc pallor were included in the
characterisation of typical retinal features of RP. It was assumed
that the mode of inheritance was autosomal dominant
when a patient appeared in every generation of the
pedigree, and when affected fathers and mothers transmitted
the phenotype to both sons and daughters. Autosomal
recessive patterns were suspected when a phenotype
appeared in the male and female progeny of unaffected
parents. X-linked recessive inheritance was suspected
when affected males passed the condition on to all of their
daughters, but to none of their sons, and female patients
married to unaffected males passed the condition on to
half of their sons and daughters. Written informed
consent was obtained from all patients before they were
enrolled in the study. This study followed the tenets of the
declaration of Helsinki (Edinburgh, 2000) and was
approved by the Institutional Review Board of Seoul
National University Hospital.
Targeted exome sequencing of RP-related genes
Customised baits were designed to capture all exons of
the 53 genes known to be associated with RP at the
time of panel design. (Table 3, Roche NimbleGen Inc,
Madison, WI). Genomic DNA from 62 patients in total
was extracted from peripheral blood as described
previously  and was sequenced using the Genome
Analyzer II. Sequencing reads were aligned to the
UCSC hg19 reference genome using BWA-0.6.1 with
default settings. Duplications were removed via Picard
v1.93, and local realignment was done by GATK v2.4-7.
Variants were identified by the Unified Genotyper from
GATK for the SNVs and indels. ANNOVAR was used to
annotate the variants. Coverage of TES data was
calculated by the ‘Depth of Coverage’ module from GATK.
Sanger sequencing for candidate variants was performed
using specific primers for each exon as demonstrated.
Prioritization of variants
A flowchart for candidate causal variant detection is
shown in Fig. 1. Exonic and splicing variants were first
selected if they had an allele frequency below 1 %
reported in the NHLBI-ESP 6500 (evs.gs.washington.edu),
1000 Genome Project (www.1000genomes.org), and an
in-house database with exomes of 192 Korean
individuals. Variants with a quality score below 20 were
excluded. Candidate variants were confirmed by Sanger
sequencing and co-segregation analysis was performed
in cases of familial RP. The potential pathogenicity of
variants was categorised into four classes (Table 4).
Briefly, Class I included pathogenic variants previously
known to cause RP, and Class II variants were expected
to cause severe damage to protein structure via
frameshift, nonsense, and missense variants, which were likely
to cause severe functional change via Polyphen 2 ,
SIFT , and MutPred . Class III included variants
least likely to be causative and consisted of missense
variations that were predicted to be benign or tolerable.
All other types of variants were categorised as Class IV.
Novel nonsynonymous variants were assumed to be
possibly pathogenic if the variant was predicted to be
pathogenic by at least two of the three methods. Both
probably and possibly damaging mutations were
classified as suspected pathogenic variants by Polyphen2.
With regard to SIFT, damaging mutations were classified
as pathogenic. For Mutpred, a general score higher than
0.5 was categorised as possibly pathogenic.
Additional file 1: Table S1. Quality of sequencing results. Table S2.
Clinical features of 10 familial RP cases whose strong variants were detected
by targeted resequencing. Table S3. Clinical features of 7 sporadic RP cases
whose strong variants were detected by targeted resequencing. Table S4.
In silico prediction for nonsysnonymous variants. Figure S1. Pedigrees of 10
familial cases whose strong variants were detected by targeted re-sequencing.
Figure S2. Fundus photograph and optical coherence tomography of the
The authors declare that they have no competing interests.
CKY participated in sequence alignment and clinical assessment and drafted
the manuscript. JYS and JHP participated in clinical analysis and helped to
draft the manuscript. NKDK and JGJ participated in bioinformatics analysis of
TES data and wrote the manuscript. HHE and HL participated in sequence
alignment and Sanger sequencing. WYP and HGY conceived the study and
participated in its design and coordination, as well as helping to draft the
manuscript. All authors read and approved the final manuscript.
This study was supported by a grant of the Korea Health Technology R&D
Project through the Korea Health Industry Development Institute (KHIDI),
funded by the Ministry of Health & Welfare, Republic of Korea
(HT12C001420014) and Samsung Medical Center grant.
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