Exome Sequencing Identifies Compound Heterozygous Mutations in CYP4V2 in a Pedigree with Retinitis Pigmentosa
et al. (2012) Exome Sequencing Identifies Compound Heterozygous Mutations in CYP4V2 in a Pedigree with
Retinitis Pigmentosa. PLoS ONE 7(5): e33673. doi:10.1371/journal.pone.0033673
Exome Sequencing Identifies Compound Heterozygous Mutations in CYP4V2 in a Pedigree with Retinitis Pigmentosa
Yun Wang 0
Liheng Guo 0
Su-Ping Cai 0
Meizhi Dai 0
Qiaona Yang 0
Wenhan Yu 0
Naihong Yan 0
Xiaomin Zhou 0
Jin Fu 0
Xinwu Guo 0
Pengfei Han 0
Jun Wang 0
Xuyang Liu 0
Andreas R. Janecke, Innsbruck Medical University, Austria
0 1 Ophthalmic Laboratories & Department of Ophthalmology, State Key Laboratory of Biotherapy, Translational Neuroscience Center, West China Hospital, Sichuan University , Chengdu, Sichuan Province , People's Republic of China, 2 Shenzhen Eye Hospital, Jinan University , Shenzhen, People's Republic of China, 3 BGI-Shenzhen, Shenzhen, Guangdong Province , People's Republic of China
Retinitis pigmentosa (RP) is a heterogeneous group of progressive retinal degenerations characterized by pigmentation and atrophy in the mid-periphery of the retina. Twenty two subjects from a four-generation Chinese family with RP and thin cornea, congenital cataract and high myopia is reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family presented with bone spicule-shaped pigment deposits in retina, retinal vascular attenuation, retinal and choroidal dystrophy, as well as punctate opacity of the lens, reduced cornea thickness and high myopia. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. After mutation analysis in a few known RP candidate genes, exome sequencing was used to analyze the exomes of 3 patients III2, III4, III6 and the unaffected mother II2. A total of 34,693 variations shared by 3 patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in the rest family members by PCR and Sanger sequencing. Compound heterozygous c.802-8_810del17insGC and c.1091-2A.G mutations of the CYP4V2 gene, known as genetic defects for Bietti crystalline corneoretinal dystrophy, were identified as causative mutations for RP of this family.
Funding: This work was supported by grants from the National Natural Science Foundation of China (NSFC 81000370) and the National Basic Research Program
of China (973 Program, 2011CB510201). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Retinitis pigmentosa (RP) is a heterogeneous group of
progressive retinal degenerations characterized typically by pigmentation
and atrophy in the mid-periphery of the retina. It was estimated to
affect 1 in 3500 in the general population [1,2]. Symptoms for RP
include night blindness, tunnel vision and bone-spicule
pigmentation in retina.
Considerable clinical and genetic heterogeneity was
demonstrated in RP patients, with wide variations in age of onset,
severity, clinical phenotype, rate of progression and pattern of
inheritance. Genotype-phenotype correlations are not strong
enough to predict for RP. About 2030% of patients with RP
also presented with non-ocular disorders such as hearing loss,
obesity, and cognitive impairment. Such cases fall within more
than 30 different syndromes .
Over 50 genes have been identified to cause RP, but still only
explain no more than half of the clinical cases . Therefore, there
has been limited success with approaches of screening of known
candidate genes for RP by conventional Sanger sequencing.
Fortunately, exome sequencing technique has come to the aid by
enabling the identification of disease-associated mutations by
sequencing the whole exome of a small number of affected
In the present study, disease-associated mutations were
identified in a large Chinese family with RP complicated with congenital
cataract, corneal thinning and high myopia using the exome
Materials and Methods
Subjects and Clinical Assessment
Twenty two family members underwent complete
ophthalmologic examinations, including slit-lamp biomicroscopy, fundus
examination, fundus fluorescein angiography, optical coherence
tomography (OCT) for assessment of retinal thickness, B-scan
ultrasonagraphy for detection of vitreous and retina, central
corneal thickness (CCT) and full-field flash electroretinography
(ERG). Written informed consent was obtained in accordance
with the Declaration of Helsinki before blood samples were
taken for analysis (see attachment for details). The study was
approved by West China Hospital, Sichuan University Institute
Venous blood samples were obtained from twenty two family
members in EDTA Vacutainers. Genomic DNA was extracted
from 200 ml peripheral venous blood using Qiamp Blood DNA
mini Kit (Qiagen, Hilden, Germany) according to the
manufacturers instructions. DNA samples were stored at 220uC until
used. DNA integrity was evaluated by 1% agarose gel
Polymerase chain reaction (PCR)-based, direct sequencing was
used in the analysis. A number of candidate genes previously
shown to be mutated in RP patients including RP1, RP2, RPGR,
RHO, RDS, ROM1, TULP1 and RPE65 were sequenced. These
genes were known to be frequently involved in autosomal
dominant, recessive or X-linked RP [3,7]. Intronic primers
flanking the exons of the candidate genes were designed based
on gene sequences of RP1 (GenBank NG_009840.1), RP2
(NG_009107.1), RPGR (NG_009553.1), RHO (NG_009115.1),
RDS (NG_009176.1), ROM1 (NG_009845.1), TULP1
(NG_009077.1) and RPE65 (NG_008472.1) synthesized by
BGIBeijing, Beijing, China. DNA fragments were then amplified by
PCR using a MyCycler thermocycler (Bio-Rad, Hercules, CA)
under the following conditions: 1 ml dNTP (2 mmol/L), 5 ml 106
buffer (containing MgCl2, 210 mmol/L), 0.5 ml primer (20 pmol/
ml), 3 ml polymerase (5 U/ml) and 5 ml genomic DNA (70 ng/ml).
An aliquot of 5 ml of PCR product was subjected to electrophoresis
on 1.5% agarose gel to confirm successful DNA amplification.
Purified PCR products were directly sequenced using an ABI
377XL automated DNA sequencer (Applied Biosystems, Foster
City, CA). Sequence data were compared pair-wisely with the
related Human Genome database.
The exome sequencing was employed in this study to identify
the disease-associated genes based on the following reasons. Firstly,
given the fact that the father II1 was deceased 20 years ago and his
affected status cannot be ascertained, the exact inheritance pattern
cannot be decided with certainty. Secondly, undertaking Sanger
sequencing of further RP-associated genes would not be
costeffective. Thirdly, the condition in this family might be due to
mutations in a gene not previously reported to be associated with
Exome sequencing was performed on 3 patients (III2, III4, and
III6) and II2 (the mother of all the patients) by BGI Inc.,
Shenzhen, China. The reason for choosing the mother (II2) was
that, the data from her was essentially needed in almost all
inheritance models including the autosomal recessive model, in
which the mother was a carrier. Thirty mg human genomic DNA
was extracted from peripheral venous blood samples of each
participant. Agilent SureSelect target enrichment system (44 Mb)
was used to collect the protein coding regions of human genome
DNA. It covered 18134 genes in the Consensus Coding Sequence
Region database 2008(http://www.ncbi.nlm.nih.gov/projects/
CCDS/). The qualified genomic DNA samples were randomly
fragmented on a Covaris Acoustic System, before adapters were
ligated to both ends of the resulting fragments. The adapter-ligated
templates were purified by Agencourt AMPure SPRI beads.
Fragments with insert size about 250 bp were excised. Extracted
DNA was amplified by ligation-mediated PCR (LM-PCR),
purified, and hybridized to SureSelect Biotinylated RNA Library
(BAITS) for enrichment. Hybridized fragments were bound to the
strepavidin beads, whereas non-hybridized fragments were washed
out after 24 h. Captured LM-PCR products were subjected to
Agilent 2100 Bioanalyzer to estimate the magnitude of
enrichment. Each captured library was then loaded on HiSeq 2000
platform for sequencing. Each captured library was sequenced
independently to ensure each sample had at least 30-fold coverage.
Raw image files were processed by Illumina Pipeline v1.7 for
basecalling with default parameters and the sequences of each
individual were generated as 90 bp paired-end reads. We obtained
a mean exome coverage of 466, which provided sufficient depth
to accurately call variants at ,96% of each targeted exome.
The sequencing reads were aligned to the human reference
genome (NCBI Build 36.3) with SOAPaligner (soap2.21) .
Based on the SOAP alignment results, the software SOAPsnp v
1.05  was used to assemble the consensus sequence and call
genotypes in target regions. Data were provided as lists of
sequence variants (SNPs and short indels) relative to the reference
genome. Identified variants were filtered against the Single
Nucleotide Polymorphism database (dbSNP 129, http://www.
ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), 1000 genome
project (www.1000genomes.org/,1094 individuals from the
20101123 sequence and alignment release of the 1000 genomes
project), HapMap 8(http://hapmap.ncbi.nlm.nih.gov/) database
and YH database  (Table 1 and Table 2).
We collected reads that were aligned to the designed target
regions for SNP identification and subsequent analysis. The
consensus sequence and quality of each allele was calculated by
SOAPsnp. We filter SOAPsnp results as follows: Base quality is
more than 20, depth is between 4 and 200, estimate copy number
is equal or less than 2 and the distance between two SNPs must be
longer than 4.
Verification of Variants
Sanger sequencing was used to determine whether any of the
remaining variants co-segregated with the disease phenotype in
this family. Primers flanking the candidate loci were designed
based on genomic sequences of Human Genome (hg18/build36.3)
and synthesized by BGI-Beijing, Beijing, China. All shared
variants of the three affected individuals after filtering were then
confirmed by direct polymerase chain reaction (PCR) and
analyzed on an ABI 3730XL Genetic Analyzer. Sequencing data
were compared pair-wisely with the Human Genome database.
Clinical Assessment and Findings
A four-generation family from Sichuan Province of China was
recruited in this study (Figure 1). Ophthalmic examinations
identified 4 affected individuals as RP patients among the 22
examined family members.
Affected members of this family exhibited similar clinical
features. They suffered from high myopia since about 10 years
old. Visual acuity dropped progressively to light perception in
their 50 s. Fundus examination and fluorescein angiography in
affected patients demonstrated peripheral pigmentation, retinal
choroidal atrophy and retinal vascular attenuation in the retina
(Figure 2A, 2B). OCT scan demonstrated retinal atrophy
(Figure 2C). ERG records showed no detectable cone or rod
responses in the patients (Figure 2D). These were consistent with
the diagnosis of RP. Punctate opacities of the lens were revealed in
affected members under slit-lamp examination (Figure 2E).
Corneas of affected members were also found to be thinner.
CCT of the unaffected were above 500 mm, while CCT of the
patients was in range 460-475 mm on average (Except IV7, who
underwent LASIK surgery) (Table 3). B-scan ultrasonagraphy
showed posterior scleral staphyloma in all of the patients
(Figure 2F), indicating high myopia.
Direct sequencing of the RHO, RDS, RP1, RP2, RPGR (including
ORF15), ROM1, RPE65 and TULP1 exons showed no pathogenic
mutations in any of the affected individuals in this family. The
following SNPs (rs444772, rs446227, rs414352 of RP1; rs7764439,
rs390659, rs425876, rs434102 of RDS; rs5918520 of RPGR) were
found in both affected and unaffected members of this family and
were shown to have no correlations with the disease.
Exome sequencing identified 32216 SNPs and 2477 Indels that
were shared by the 3 patients. The results were then filtered
against several public variation databases, removing all previously
reported variants (Table 1, 2). Filtering all exomes for a
homozygous mutation causing the disease in the affected sibs
(III2, III4, III6), and which was present in heterozygous form in
the unaffected mother (II2, carrier), Variants satisfying a
recessive homozygous inheritance model were not identified. This
led us to investigate the possibility of recessive compound
heterozygous inheritance. Under the hypothesis of a
compoundheterozygous model, we filtered all exomes for variants present in
the heterozygous state in all affected individuals for variants and
also not present heterozygous in their mothers exome. It restricted
the results to 26 heterozygous variants (Table 4). Heterozygous
CYP4V2 c.1091-2A.G was one of the 26 variants, and was
known to be responsible for recessive BCD. The mutation was
predicted to disrupt the splicing of intron 8, resulting in an
inframe skipping of 45 amino acidencoding exon 9 .
As one heterozygous variation was identified from the father
side, the other one inherited from the mother (II2) was identified
by re-filtered the exome sequencing data for CYP4V2 variations
Table 2. Number of candidate Indels filtered against several
public variation databases.
present in all affected individuals and their mother (Table 4).
Thirteen variants of the CYP4V2 gene were identified, including
two non-synonymous variants c.775C.A and
c.802-8_810del17insGC. The former was non-pathogenic , whereas the
latter harbored a 17 bp deletion including the exon 7
spliceacceptor site, leading to an in-frame deletion of 62 amino
acidencoding exon 7 [13,14].
All the family members were then screened by PCR
amplification and Sanger sequencing for these two mutations,
c.8028_810del17insGC and c.1091-2A.G. Only patients were found
to carry both mutations (Figure 3). Phenotypes and underlying
mutations of related family members were summarized in Table 3.
In 2004, CYP4V2 defects were identified previously as causative
mutations for BCD . The same mutations found in this study
have been reported to be associated with an autosomal recessive
BCD, which exhibited a totally different phenotype from this
pedigree . It is the first time, to the best of our knowledge, to
show that mutations in CYP4V2 caused not only BCD, but also
BCD is an autosomal recessive retinal degeneration
characterized by multiple tiny glistening crystalline deposits scattered over
the fundus. The small glistening crystals can also occur in the
corneal limbus and circulating lymphocytes [11,12,15]. The
molecular basis for BCD remains unclear. Previous studies showed
that defects in lipid metabolism were associated with this disease.
In BCD patients, the level of polyunsaturated fatty acids (PUFAs)
decreased due to the abnormal metabolism of fatty acid
precursors, possibly because of the presence of the abnormal
lipidbinding protein and enzymes essentially needed in elongation and
desaturation of fatty acid [16,17].
The CYP4V2 gene encodes a member of the cytochrome P450
hemethiolate protein superfamily which is involved in oxidizing
various substrates in the metabolic pathway. The CYP4 family is
associated with endogenous fatty acid metabolism, with CYP4V2
capabling of hydroxylating the omega-3 PUFAs, including
docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)
. PUFAs are highly enriched in the brain and eye, particularly
in the retina , playing an important role in regenerating disk
membranes of the outer segments of photoreceptor cells .
Phenotypically, the patients in this family showed remarkable
differences from BCD patients who carried exactly the same
mutations . Instead of glistening crystalline deposits, pigment
deposits, retinal vascular attenuation and choroidal atrophy were
the most significant observations in the fundus. In addition, the
patients in our study had younger average age at onset and worse
visual acuity than those reported . Interestingly, abnormalities
in lipid metabolism was also noticed in RP patients . For
example, serum DHA was lower in patients with RP .
DHA deficiency may affect the activity of omega-3 fatty acid
desaturation and elongation reactions, and then alter the physical
and functional properties of outer segment membranes. Animal
studies have shown that reduction of DHA in dietary intake results
in abnormal ERGs and visual loss [25,26]. Clinical trial in RP
patients showed that progression of RP could be prevented or
slowed down when the patients were treated with DHA .
Dietary supplementation of DHA in such patients would by-pass
some biosynthetic and transport steps and may restore blood levels
of DHA back to normal . All these suggest a link between
DHA deficiency and risk of RP, and between CYP4V2 defects and
the pathogenesis of RP.
Since the inheritance pattern of this pedigree was not clearly
clarified, making the genetic analysis of this pedigree difficult. We
presumed autosomal recessive as the most likely inheritance
model. Mutational screening for several genes associated with
autosomal recessive inheritance failed to identify the causative
gene(s). Given the fact that many mutations in at least 50 genes
are known to cause autosomal recessive RP (RetNet: http://www.
7 A 4
5 A 5
8 M 7
Exome sequence variants shared
by all affected individuals
Presented heterozygous Presented heterozygous in carrier
in carrier (II2) (II2)
sph.uth.tmc.edu/Retnet/sum-dis.htm) and more to be identified,
exome sequencing was employed for genetic analysis of this
pedigree. Our results showed that this approach can be used to
effectively narrow down candidate genes and to identify genetic
defects responsible for Mendelian-inheritance diseases in
The mother II2 in this study was not a real negative control for
exome sequencing since she was supposed to be a carrier in the
autosomal recessive model. Initial analysis of exome sequencing
showed that c.1091-2A.G in CYP4V2 was first carried by the 3
patients (III2, III4, III6); further sequence verification showed that
this variation was present not only in another patient (III8), but
also in unaffected individuals, including II1s brother (II3). It was
thus presumed that this heterozygous variation was inherited from
father II1, and carrying this variation only was not pathogenic. In
a compound-heterozygous model, as one heterozygous variation
was identified from the father side (II3), the other one inherited
from mother (II2) was identified by re-filtered the exome
sequencing data for variations present in all affected individuals
and their mother. The mutation c.802-8_810del17insGC in
CYP4V2 was then identified, since only the four patients carried
both c.1091-2A.G and c.802-8_810del17insGC in CYP4V2.
Among the mutations identified in this pedigree, c.1091-2A.G
of CYP4V2 was predicted to disrupt the splicing of intron 8,
resulting in an in-frame skipping of 45-amino-acid encoding exon
9. [11,12] The other 39 splicing acceptor site mutation,
c.8028_810del17insGC was reported as a frequent founder mutation in
East Asian populations [13,29]. The change in this splicing
acceptor site was expected to cause an in-frame deletion of 62
amino acid-encoding exon 7, which was confirmed by reverse
transcriptase (RT)-PCR [13,14].
In summary, a RP- associated gene, CYP4V2, was identified by
exome sequencing. The phenotypegenotype correlations with
regard to CYP4V2 sequence alterations were discussed. Our study
highlights the clinical heterogeneity of RP and demonstrates that
exome sequencing can be a valuable method to the diagnosis of
genetic diseases. Most interestingly, the same compound
heterozygous mutations were identified to cause two retinal disorders
with totally different phenotypes. The underlying mechanisms
need to be further elucidated.
We thank the family for their cooperation and continued interest.
Conceived and designed the experiments: XL JW. Performed the
experiments: YW LG. Analyzed the data: MD XG PH JF. Contributed
reagents/materials/analysis tools: XZ QY WY NY. Wrote the paper: YW
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