Satoshi Narumi,1Chikahiko Numakura,2Takashi Shiihara,2,3Chizuru Seiwa,4Yasuyuki Nozaki,5
Takanori Yamagata,5Mariko Y. Momoi,5Yoriko Watanabe,6Makoto Yoshino,6Toyojiro Matsuishi,6
Eriko Nishi,7Hiroshi Kawame,7Tsutomu Akahane,8Gen Nishimura,9Mitsuru Emi,10
and Tomonobu Hasegawa1*
1Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan
2Department of Pediatrics, Yamagata University School of Medicine, Yamagata, Japan
3Division of Neurology, Gunma Children’s Medical Center, Shibukawa, Japan
4Department of Pediatrics, Yamagata Prefectural Comprehensive Rehabilitation and Education Center, Yamagata, Japan
5Department of Pediatrics, Jichi Medical University, Tochigi, Japan
6Department of Pediatrics, Kurume Graduate School of Medicine, Kurume, Japan
7Division of Medical Genetics, Nagano Children’s Hospital, Nagano, Japan
8Department of Orthopedic Surgery, Nagano National Hospital, Nagano, Japan
9Department of Radiology, Tokyo Metropolitan Kiyose Children’s Hospital, Tokyo, Japan
10DNA Chip Research, Inc., Yokohama, Japan
Received 28 May 2009; Accepted 2 October 2009
Osteoporosis-pseudoglioma syndrome (OPS; OMIM 259770) is
an autosomal-recessive genetic disorder characterized by severe
osteoporosis and visual disturbance from childhood. Biallelic
mutations in the low-density lipoprotein receptor-related pro-
tein 5 gene (LRP5) have been frequently detected, while a subset
the clinical and molecular findings of four unrelated Japanese
and ocular phenotypes of OPS, namely severe juvenile osteopo-
rosis and early-onset visual disturbance, with or without mental
retardation. We undertook standard PCR-based sequencing for
LRP5 and found four missense mutations (p.L145F, p.T244M,
one splice site mutation (c.1584þ1G>A) among four OPS pa-
one had only one heterozygous splice site mutation. In this
patient, RT-PCR from lymphocytic RNA demonstrated splice
error resulting in 63-bp insertion between exons 7 and 8.
Furthermore, the patient was found to have only mutated
RT-PCR fragment, implying that a seemingly normal allele
did not express LRP5 mRNA. We then conducted custom-
designed oligonucleotide tiling microarray analyses targeted to
microdeletion encompassing exons 22 and 23 of LRP5. We
found various types of LRP5 mutations, including an exon-level
Tomonobu Hasegawa, M.D., Ph.D., Department of Pediatrics, Keio
University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo
160-8582, Japan. E-mail: email@example.com
Published online 22 December 2009 in Wiley InterScience
How to Cite this Article:
Narumi S, Numakura C, Shiihara T, Seiwa C,
Nozaki Y, Yamagata T, Momoi MY,
Watanabe Y, Yoshino M, Matsuishi T, Nishi
E, Kawame H, Akahane T, Nishimura G, Emi
M, Hasegawa T. 2010. Various types of LRP5
pseudoglioma syndrome: Identification of a
7.2-kb microdeletion using oligonucleotide
Am J Med Genet Part A 152A:133–140.
? 2009 Wiley-Liss, Inc.
deletion that is undetectable by standard PCR-based mutation
screening. Oligonucleotide tiling microarray seems to be a
powerful tool in identifying cryptic structural mutations.
? 2009 Wiley-Liss, Inc.
Key words: osteoporosis-pseudoglioma syndrome; low-density
lipoprotein receptor-related protein 5 (LRP5); mutation; micro-
deletion; comparative genomic hybridization
porosis is characterized by low bone mineral density (BMD) and
bone microarchitectural alterations. Because BMD is highly heri-
table trait [heritability estimates, 0.6–0.8, Cummings and Melton,
2002], elucidating the genetic regulation of BMD has a crucial role
in unveiling molecular mechanisms of osteoporosis.
There are a handful of genetic forms of osteoporosis showing
Mendelianinheritance, whichusually onset earlierand cause more
profound bone fragility. These syndromes are rare but have pro-
doglioma syndrome (OPS;OMIM259770)is anexample of such a
syndromic osteoporosis. OPS is usually associated with biallelic
inactivating mutations in the low-density lipoprotein receptor-
related protein 5 gene [LRP5; Gong et al., 2001], of which gene
[Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000].
Clinically, OPS is hallmarked by severe osteoporosis and visual
disturbance from childhood [Frontali et al., 1985]. Osteoporosis
typically onsets in juvenile period and results in long-bone frac-
extremities, and short stature. Congenital or early-onset visual
disturbance arises from the degeneration of vitreoretinal tissue
and manifests a wide range of ophthalmologic problems including
phthisis bulbi, retinal detachment, falciform folds, and micro-
phthalmia. Of interest, inactivating LRP5 mutations is also associ-
ated with a genetic ophthalmologic disorder named familial
exudative vitreoretinopathy (FEVR; MIM 601813) [Jiao et al.,
2004; Toomes et al., 2004]. Some FEVR patients were confirmed
variation of BMD among general population [Koay et al., 2004;
Mizuguchi et al., 2004; Urano et al., 2004; Koller et al., 2005; van
Meurs et al., 2006; Richards et al., 2008].
is growing, understanding of molecular genetics of OPS remains
still incomplete. In the most intensive clinical/genetic study that
enrolled 37 OPS patients, 26 patients were shown to have biallelic
LRP5 mutations but the remaining 11 patients were shown to have
only one (n¼4) or no mutation (n¼7) [Ai et al., 2005]. Thus,
roughly 30% of OPS patients cannot be explained by the current
genetic model. Here, we report on the clinical and molecular
findings of four unrelated Japanese patients with OPS. Standard
PCR-based sequencing revealed that three patients had biallelic
heterozygous LRP5 mutations, but one had only one heterozygous
a seemingly normal allele of the patient did not express LRP5
mRNA. We then conducted custom-designed oligonucleotide
tiling microarray analyses targeted to a 600-kb genome region
harboring LRP5 and identified a novel 7.2-kb microdeletion en-
compassing exons 22 and 23 of LRP5. We exemplify the usefulness
of oligonucleotide tiling microarray in detecting cryptic structural
mutations that are undetectable by standard PCR-based mutation
Approval for this study was obtained from the institutional review
board of Keio University School of Medicine. We obtained written
informed consent for molecular studies from the patients and/or
consanguinity was reported among the parents. Clinical informa-
tion is summarized in Table I.
Patient 1, an 18-year-old boy, was born at term after uneventful
pregnancy and delivery. Reportedly, he could not follow objects
well with eyes in his early infancy. Ophthalmologic examination
revealed microphthalmia, cataract, and retinal detachment in both
He had moderate mental retardation: intelligence quotient (IQ)
score on the Wechsler Intelligence Scale for Children was 45 at age
11 years. Between ages 7 and 13 years, he had three fractures in the
distal diaphysis of the right femur caused by low-impact trauma.
-matched reference by Nishiyama et al., 1999] at age 14 years.
Patient 2, a 23-year-old male, was born at term after uneventful
pregnancy and delivery. At age 1 month, his parents noticed
leukocoria in his left eye. Ophthalmologic examination revealed
microphthalmia and retinal detachment in the left eye and persis-
tent hyperplasia of the primary vitreous in the right eye. His visual
acuity was 0.06 right and hand motion left at last visit. He had no
trauma. At age 9 years, he had compression fractures in his spines
(Th7-10, L1) with no recognizable cause. Osteopenia, kyphosco-
liosis, and platyspondyly in all vertebrae were shown by radio-
graphs. At age 11 years, he became wheelchair dependent due to
multiple compression fractures. He had short stature with adult
height of 151.6cm (?3.2 SD). The BMD of the lumber spine was
0.47g/cm2(?5.6 SD) at age 13 years.
Patient 3, a 10-year-old male, was born at term after uneventful
vitreous body in the right eye. In the left eye, insufficient develop-
ment of peripheral retinal vessel, abnormal vascular growth, and
retardation: his verbal IQ score on the Wechsler Intelligence Scale
134AMERICAN JOURNAL OF MEDICAL GENETICS PART A
TABLE I. Clinical Phenotypes of LRP5 Mutation Carriers
PHPV, retinal detachment,
PHPV, retinal detachment
Mental retardation, muscular
hypotonia, joint laxity
PHPV, retinal detachment,
Ebstein anomaly of heart
BMD, bone mineral density; NA, not available; OPS, osteoporosis-pseudoglioma syndrome; PHPV, persistent hyperplasia of primary vitreous.
Abnormal values are highlighted in bold.
aMeasured after 2 years of oral bisphosphonate therapy. Data before therapy are unavailable.
bRoutine fundoscopic examination was normal in these subjects.
cMeasured after three courses of intravenous bisphosphonate therapy. Sex- and age-matched reference data are unavailable for pretreatment age.
dAll fractures were caused by moderate-to-severe-impact trauma (e.g., fall down during skiing).
NARUMI ET AL.
down while skiing resulting in a sacral fracture. No other fractures
have been reported. Physical examination at age 10 years showed
joint laxity, muscular hypotonia, and increased magnitude of
patellar tendon reflex. Skeletal radiographs revealed osteopenia
andplatyspondyly in allvertebrae. Longbones werenot deformed.
The lumber spine BMD was0.17g/cm2(?11.2 SD) at age 10 years.
minor breathing problems. At age 1 month, his parents noticed
bilateral leukocorea. Subsequent examination revealed retinal de-
as having bilateral persistent hyperplasia of the primary vitreous.
moderate mental retardation: Japanese Developmental Scale for
visually disabled children was 38 at age 3 years. Between ages 3 and
6 years, he had five peripheral fractures (at his femur, wrist, and
radiographs at age 5 years showed marked osteopenia and platy-
spondyly in all vertebrae. The thorax was narrow with slender
posterior ribs. Long bones were slender and deformed mildly. He
had short stature (height: 100.0cm at age 5 years; ?2.3 SD). The
lumber spine BMD was 0.19g/cm2that seemed extremely low (no
age-matched reference for the Japanese is available).
MATERIALS AND METHODS
PCR-Based Mutation Screening
We extracted genomic DNA from peripheral blood of the four
probands (and family members of each proband if available) by a
standard technique. We analyzed 23 coding exons and flanking
introns of LRP5 by PCR and direct sequencing. Primer sequences
and PCR conditions are available on request. The sequence was
determined by a BigDye Dideoxy Sequence Kit (Applied Biosys-
tems, Foster City, CA) and an automated sequencer ABI3130xl
(Applied Biosystems). Detected mutations were tested in 100
Japanese control individuals.
We isolated total RNA from peripheral lymphocytes of Patient 4
and his parents using a PAXgene Blood RNA Kit (Qiagen, Hilden,
Germany). Lymphocytic cDNA was synthesized by reverse tran-
scriptase reaction with oligo-dT primer (SuperScript III; Invitro-
gen, Carlsbad, CA). Fragments spanning exons 6–8 was PCR
amplified and sequenced using following primers: 50-ATT GCC
ATC GAC TAC GAC CCG C-30and 50-GCA GCT GGT CAA TGA
TGA CGT CC-30.
DNA samples from Patient 4, the father of Patient 4, and two
control individuals were subject to high-density oligonucleotide-
fabricated a custom-designed microarray targeted to a 600-kb
67,600,000–68,200,000 [NCBI Build 36.1, hg18]) according to
the methods previously described [Barrett et al., 2004; Perry
et al., 2008]. In brief, we used Agilent website (http://
earray.chem.agilent.com/earray/) to select and design our custom
size (Agilent Technologies, Santa Clara, CA), with median spacing
manufacturer’s instructions [de Smith et al., 2007]. In brief, test
and reference (NA19000, a Japanese male from HapMap project)
Cy5 (test) and Cy3 (reference) with a ULS Labeling Kit (Agilent
Technologies). For each sample, respective labeling reactions were
mixed and then separated prior to hybridizing to each of the two
preannealed with Cot-1 DNA (Invitrogen) and blocking reagent
(Agilent Technologies), and then hybridized to the arrays for 40hr
hybridization and washes, the arrays were scanned at 5mm resolu-
tion with an Agilent G2505B scanner. Images were analyzed with
Feature Extraction Software 18.104.22.168 (Agilent Technologies), with
the CGH-v4-95-Feb07 protocol for background subtraction and
Determination of the Deletion Breakpoints
APCRprimerpair whoseforwardprimer(50-CACCCT TTC TCT
CCA CCT GTC TAA T-30) and reverse primer (50-GGT CTT CCA
TCC CTT CTT TTA GTG A-30) located in intron 21 and 1.5-kb
downstream of exon 23, respectively, was used to amplify and
the deletion involving exons 22 and 23.
Using standard PCR-based sequencing, we detected three novel
LRP5 mutations (c.1145C>T, p.P382L; c.1584þ1G>A [splice
donor site in intron 7]; and c.1655C>T, p.T552M) and three
previously described mutations [c.433C>T, p.L145F, Qin et al.,
2005; c.731C>T, p.T244M, Ai et al., 2005; and c.4600C>T,
p.R1534X, Gong et al., 2001] in the four OPS patients (Fig. 1A).
These mutations were not found in 100 control individuals. The
residues affected by missense mutations (Leu145, Thr244, Pro382,
and Thr552) were conserved throughout vertebrate evolution
(Fig. 1B). Three patients had two heterozygous mutations:
p.[T244M]þ[P382L] for Patient 1, p.[L145F(þ)T552M] for
Patient 2, and p.[P382L]þ[R1534X] for Patient 3 (Fig. 2). Patient
4 had only one heterozygous splice site mutation (c.1584þ1G>A)
that was transmitted from the mother.
Genetic analyses for family members revealed seven subjects
subjects are summarized in Table I.
To test the effect of c.1584þ1G>A on splicing of exon 7, we
performed RT-PCR spanning exons 6–8 from lymphocytic cDNA
of Patient 4 and the mother. Gel electrophoresis of the PCR
products showed a larger fragment than expected in the two
subjects (Fig. 3A). Moreover, we unexpectedly found that Patient
136AMERICAN JOURNAL OF MEDICAL GENETICS PART A
4 had only mutated RT-PCR fragment, while the mother had both
wild type and mutated fragments. Sequencing of the mutant
fragment revealed a 63-bp insertion between exons 7 and 8. The
inserted sequence was identical to 5’ terminal of intron 7 (data not
shown), indicating cryptic splice donor site utilization at 63-bp
downstream of the native exon/intron junction. This mutation is
predicted to add extra 21 amino acids without termination codon
after Glu528 (p.E528_V529ins21).
Identification of a 7.2-kb Microdeletion
We hypothesized that a seemingly normal allele of Patient 4 would
not express intact LRP5 mRNA due to yet unidentified mutation,
because only mutated RT-PCR fragment was amplified. The father
of Patient 4 had significantly low BMD but had no LRP5 mutation
in the coding regions, implying that the cryptic mutation was
transmitted from the father. Candidatesfor such mutation include
a regulatory mutation (e.g., deletion of enhancer element) and a
submicroscopic structural mutation (e.g., exon-level deletion/
these types of mutation, we conducted custom-designed oligo-
nucleotide tiling aCGH analyses targeted to a 600-kb genomic
region harboring LRP5. As a result, we discovered a novel 7.2-kb
FIG. 1. IdentificationofsixpointmutationsinLRP5.A:Partialsequences
methionine in place of Thr552, and stop codon (TGA) in place of
letter amino acid ClustalW alignments of residues surrounding the
four missense mutations. The mutated residues are shaded in the
species in which the residue is evolutionarily conserved. Blue,
negatively charged residues; red, positively charged residues; gold,
hydrophobic residues; black, other residues.
FIG. 2. Pedigrees of the four patients with osteoporosis-
pseudoglioma syndrome. Family studies revealed a total of eight
individuals having a monoallelic LRP5 mutation. The genotype of
thefather ofPatient2isinferred (indicated bybracket), whereas
all others have been determined by molecular analysis. Clinical
phenotypes of the monoallelic mutation carriers are summarized
in Table I.
NARUMI ET AL.
microdeletion encompassing exons 22 and 23 (Fig. 3B; designated
g.Ex22_Ex23del in this report). Sequence analysis of the centro-
present in the AluS and AluJ regions, respectively, showing 83%
67974774)del](NCBI Build 36.1, hg18) (Fig. 3C). This Alu–Alu
recombination indicates an unequal crossing-over in the father or
anancestor. Thisdeletion wasabsent inthe mother,sister,and 100
We have reported on four OPS patients with biallelic LRP5 muta-
tions. Various types of mutations were observed, including four
missense mutations (p.L145F, p.T244M, p.P382L, and p.T552M),
one nonsense mutation (p.R1534X), one splice site mutation
(c.1584þ1G>A), and one multi-exon deletion (g.Ex22_Ex23del).
The p.L145F has been described in a Japanese patient with
autosomal-dominant FEVR with osteopenia [Qin et al., 2005] but
has not been observed among OPS patients. To our knowledge,
p.L145F is the first mutation that causes both OPS and FEVR. The
mother of Patient 2 was heterozygous for p.L145F and had low
BMD, but had no ocular phenotype (Table I). It is not surprising,
because the majority of family members of OPS patients
(monoallelic LRP5 mutation carriers) did not have ocular pheno-
type in the previous large-scale study [Ai et al., 2005]. Low pene-
trance of the ocular phenotype in monoalleic mutation carriers
would explain those observations. Phenotype of FEVR is generally
variable [Warden et al., 2007], although no data taking individual
genotypes into account have been available so far.
Two novel missense mutations, namely p.P382L and p.T552M,
are located in the second YWTD-EGF-like domain. Although we
of them, we believe that these mutations are pathological, because
FIG. 3. Identification of a 7.2-kb deletion encompassing exons 22 and 23 of LRP5. A: Left panel shows an agarose gel electrophoregram of RT-PCR
(631bp). Note that Patient 4 had only mutated fragment. Right panel shows schematic diagram of the RT-PCR fragments. Sequencing of the
fragments revealed that the mutant had 63-bp insertion between exons 7 and 8. The inserted sequence was identical to 5’ terminal of intron 7. B:
arrowhead indicates the deleted region. C: A schematic viewof chromosome 11q13.2 (physical position, 67.96–67.98Mb;NCBI Build 36.1,hg18).
This region contains exons 19–23 of LRP5, and a total of 18 Alu repeats (red, AluY; orange, AluS; yellow, AluJ; brown, degenerated Alu). Deletion
breakpoints were determined by PCR and sequencing with a deleted region-specific primer pair.
138 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
(1) the second YWTD-EGF-like domain is the most common site
of inactivating LRP5 mutations [Ai et al., 2005; Qin et al., 2005];
(2) Pro382 and Thr552 are highly conserved residues; and
(3) structures of the side chains are altered considerably. As for
p.P382L, detection in two unrelated OPS patients is additional
The most remarkable finding of the present study is identifica-
tion of a 7.2-kb microdeletion. This mutation was undetectable by
standard PCR-based mutation screening but could be detected by
of the smallest deletions identified by aCGH-based analyses. The
deletion encompasses exons 22 and 23 of LRP5 and would impair
mRNA expression, because exon 23 (the last exon) has polyade-
nylation signal that is required for mRNA stabilization. In OPS
patients, detecting only one mutation is not uncommon [Ai et al.,
2005]. Exon-level deletion/duplication mediated by Alu–Alu re-
would be a source of cryptic mutations in a subset of patients.
Extensive screening for submicroscopic structural mutations will
be required to draw conclusions. Several methods are available. As
Chung et al. pointed, multiplex ligation-dependent probe amplifi-
cation (MLPA) is one possibility. However, although MLPA can
find exon-level dosage changes reliably, it cannot detect mutations
occurring in noncoding regions (e.g., promoter and enhancer) as
well as balanced structural mutations (e.g., inversion). As for
detectionof mutationslocated in noncoding regions, oligonucleo-
tide tiling aCGH seems to be an attractive alternative. Previous
commercially available aCGH-based approaches, including bacte-
rial artificial chromosome aCGH and genome-wide oligonucleo-
tide aCGH, have relatively low resolution (typically >100kb), and
thus are unsuitable for detecting exon-level structural changes
[Zhang et al., 2008]. This limitation can now be overcome by
custom-designed oligonucleotide tiling aCGH, which has near-
balanced structural mutations (e.g., exon-level inversion), which
are undetectable by MLPA or oligonucleotide tiling aCGH. RNA
analyses, such as RT-PCR, might detect a part of such mutations,
however, those analyses cannot detect mutations that abolish
mRNA expression (e.g., mutation accompanied by nonsense-
mediated mRNA decay). As target of genome analyses (MLPA and
oligonucleotide tiling aCGH) and RNA analyses differs, these
methods would work complimentarily in detecting cryptic
structural mutations. Therefore, detection power will be
maximized if they are applied in combination as we successfully
To conclude, we described four OPS patients who had various
types of LRP5 mutations. Our observations provide not only in-
sights into structure–function relationship of LRP5, but also ex-
using oligonucleotide tiling aCGH.
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