Osteoporos Int DOI 10.1007/s00198-015-3245-4

CASE REPORT

Potential blindness in children of patients with hereditary bone disease V. Kheir 1 & F. L. Munier 1,2 & B. Aubry-Rozier 3 & D. F. Schorderet 1,2,4

Received: 27 February 2015 / Accepted: 8 July 2015 # International Osteoporosis Foundation and National Osteoporosis Foundation 2015

Abstract Mono- and bi-allelic mutations in the low-density lipoprotein receptor related protein 5 (LRP5) may cause osteopetrosis, autosomal dominant and recessive exudative vitreoretinopathy, juvenile osteoporosis, or persistent hyperplastic primary vitreous (PHPV). We report on a child affected with PHPV and carrying compound mutations. The father carried the splice mutation and suffered from severe bone fragility since childhood. The mother carried the missense mutation without any clinical manifestations. The genetic diagnosis of their child allowed for appropriate treatment in the father and for the detection of osteopenia in the mother. Mono- and biallelic mutations in LRP5 may cause osteopetrosis, autosomal dominant and recessive exudative vitreoretinopathy, juvenile osteoporosis, or PHPV. PHPV is a component of persistent fetal vasculature of the eye, characterized by highly variable expressivity and resulting in a wide spectrum of anterior and/or posterior congenital developmental defects, which may lead to blindness. We evaluated a family diagnosed with PHPV in their only child. The child presented photophobia during the first

3 weeks of life, followed by leukocoria at 2 months of age. Molecular resequencing of NDP, FZD4, and LRP5 was performed in the child and segregation of the observed mutations in the parents. At presentation, fundus examination of the child showed a retrolental mass in the right eye. Ultrasonography revealed retinal detachment in both eyes. Thorough familial analysis revealed that the father suffered from many fractures since childhood without specific fragility bone diagnosis, treatment, or management. The mother was asymptomatic. Molecular analysis in the proband identified two mutations: a c.[2091+2T>C] splice mutation and c.[1682C>T] missense mutation. We report the case of a child affected with PHPV and carrying compound heterozygous LRP5 mutations. This genetic diagnosis allowed the clinical diagnosis of the bone problem to be made in the father, resulting in better management of the family. It also enabled preventive treatment to be prescribed for the mother and accurate genetic counseling to be provided.

Electronic supplementary material The online version of this article (doi:10.1007/s00198-015-3245-4) contains supplementary material, which is available to authorized users.

Keywords Blindness . Low-density lipoprotein receptor related protein 5 . LRP5 . Multiple fractures . Osteoporosis . Persistent hyperplastic primary vitreous (PHPV) . Pseudoglioma-osteoporosis

* D. F. Schorderet [email protected]

Introduction

1

IRO - Institute for Research in Ophthalmology, Av. du Grand-Champsec 64, 1950 Sion, Switzerland

2

Department of Ophthalmology, Jules-Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland

3

Bone and Joint Department, Center of Bone Diseases, University of Lausanne, Lausanne, Switzerland

4

Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Osteoporosis and multiple fractures in a child may be due to various conditions with primary or secondary causes [1]. While the latter may have many origins, the former is only due to a limited number of inherited diseases among which osteogenesis imperfecta (OI) is the most frequent. OI represents a large group of mostly dominant, rarely recessive, inherited disorders of collagen genes of which COL1A1 and COL1A2 are the most frequently mutated. Other conditions

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such as Marfan, homocystinuria, Hajdu-Cheney, and EhlersDanlos syndromes may also be accompanied by osteoporosis. Heterozygous mutations in the low-density lipoprotein receptor related protein 5 (LRP5) gene coding for a protein that affects bone mass accrual during growth and establishment of peak bone mass have also been implicated in osteoporosis through the canonical Wnt signaling pathway [2, 3]. Wnt proteins are a family of secreted growth factors that play a critical role in many developmental processes, including osteoblastic activity and bone formation. LRP5 functions as a coreceptor for Wnt proteins, which binds Norrin and Frizzled 4 (FZD4), a membrane protein with seven transmembrane domains, and prevents glycogen synthase kinase 3 from phosphorylating βcatenin [4]. Wnt also plays a critical role in physiological regression of hyaloid vasculature during embryogenesis. Norrin, the product of NDP, binds with high affinity to the amino-terminal cysteine-rich domain of FZD4 receptor, a Wnt receptor, and induces the activation of the classical Wnt pathway through FZD4 and LRP5 [5]. Mice lacking Fzd4 as well as Ndp–/– and Lrp5–/– mice have delayed regression of hyaloid vasculature by at least 1–2 weeks [6–8]. LRP5 signaling pathway is not only required for osteoblast proliferation and matrix bone deposition by differentiated osteoblasts but also for normal regression of fetal hyaloid vessels. In mouse, hyaloid vessels start to regress at P3 and regression is completed by P16. In Lrp5–/– mice, there is persistence of hyaloid vessels, which can still be observed at 6 months of age [6, 9]. It would seem that the lack of regression of these vessels is a consequence of a lower number of apoptotic capillary segments. Ocular macrophages are necessary for the physiological cell death that accompanies the regression of hyaloid vessels [10–12]. As the number of macrophages is no different in Lrp5–/– mice compared to wild type, a ligand of Lrp5 could be implicated in macrophage action during the regression of hyaloid vessels [6]. Heterozygous Lrp5+/– mice undergo normal hyaloid vessel regression but present a bone phenotype, while homozygous mice develop osteoporosis-pseudoglioma syndrome (OPPG) [6, 13].

Fig. 1 a RetCam and b angiography montage of the left fundus showing vascular growth arrests (white arrow heads)

A

OPPG is an autosomal recessive disease characterized by loss of vision either congenital or of early onset (pseudoglioma) and bone fragility starting during childhood. Blindness may result from various conditions such as persistent hyperplastic primary vitreous (PHPV) with retrolental fibrovascular masses, congenital retinal folds, exudative retinopathy, or phthisis. In most cases, both eyes are blind, but there are instances of one eye blind and the other severely impaired [14]. The bone phenotype consists of a very low bone mass predisposing to osteoporosis and bone fractures. OPPG is rare and has been estimated to be around 1/2,000,000 in the USA with a carrier frequency of 1/700 [14]. Almost 20 years ago, Gong et al. mapped OPPG on chromosome 11 and later, Ai et al. identified mutations in LRP5 [2, 14]. Today, more than 100 mutations have been identified [14].

Case report A first child was born after an uneventful pregnancy to parents with no history of ophthalmic disease. The child presented photophobia during the first 3 weeks of life, followed by leukocoria at 2 months at which time, he was referred to the Jules-Gonin Eye hospital. Bilateral leukocoria was noted with horizontal nystagmus and no eye fixation. Biomicroscopy examination of the anterior segment was unremarkable except for the presence of bilateral persistent tunica vasculosa lentis. Fundus examination of the left eye (OS) was possible, while a retrolental mass prevented visualization of the posterior pole in the right eye (OD). One week later, the patient developed a hemorrhage masking the optic nerve head OS (Fig. 1a). Angiography OS showed that the retinal vessels were limited to zone I only (Fig. 1b). Ultrasonography revealed retinal detachment in both eyes. Ultrasound biomicroscopy (UBM) showed a narrow anterior chamber OD. The diagnosis of bilateral severe vitreoretinal dysplasia was made. Bilateral anterior vitrectomy and lensectomy were performed in order to save the globes from enucleation. Biopsy of the retrolental mass was sent for histopathology with results consistent with

B

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PHPV. Over the next month, both eyes evolved towards phthisis bulbi and total blindness. At 3 years, molecular analysis of NDP, FZD4, and LRP5 was performed. DNA was extracted from blood leukocytes after informed consent was obtained. Direct bidirectional sequencing of all PCR-amplified coding exons and adjacent junctions was performed with the ABI Dye Terminator, version 1 (Applied Biosystems, Foster City, CA), in a final reaction volume of 10 μl, and these were electrophoresed on a 3130XL ABI genetic analyzer (Applied Biosystems). Sequence of the primers and PCR conditions is listed in Additional file 1: Table S1. The proband did not experience fractures during the first 5 years of life, but the parents recently reported a fall from a swing resulting in a broken radius bone. A dual-energy X-ray absorptiometry (DXA) was planned but could not be carried out due to over excitability of the child and the parents did not agree to anesthesia. Following the results of the mutation analysis, genetic counseling was provided. A thorough familial analysis showed no consanguinity in this couple of Swiss origin. When the parents were told that the mutation they carried could have consequences, the father remembered that he suffered from many fractures since his childhood, but no diagnosis was established, either clinically or genetically. At 11 years of age, he was diagnosed with dorso-lumbar scoliosis, severe lumbar lordosis, and moderate dorsal kyphosis. At that time, the body of L1 was already cuneiform. At 12 years, he suffered a fracture of L1, followed 1 year later by fractures of the right wrist, D10 and D12. At 15 years, he fell from a tree and fractured L1 and L2. He wore a surgical corset for 3 months. X-ray exams showed a transparent aspect of the lumbar spine, the hips, and the pelvis. DXA showed a very low Z-score at −3.8 at the spine. The diagnosis of primary osteoporosis was made. In spite of additional multiple non-trauma fractures

A

B

after puberty, reported at one per year, he was lost to followup and did not receive a correct diagnosis or treatment. Following the molecular analysis results of his son, a new DXA was performed, which revealed a T-score of −2.5 for the hip and −2.8 for the spine. He received bisphosphonate therapy with favorable evolution. No new fractures were observed in the last 3 years, and DXA results showed a significant improvement. In addition, ocular angiography showed temporal vascular abnormalities that were classified in the FEVR spectrum. The mother of the proband had an unremarkable history, with no fractures and no osteoporosis. DXA was performed after the discovery of her LRP5 mutation, which showed osteopenia with a hip and spine T-score of −1.3 and −1.5, respectively.

Results Molecular analysis of NPD and FRZD4 showed no pathogenic mutations. Analysis of LRP5 revealed two unpublished mutations, a c.[2091+2T>C] mutation affecting the canonical second base of the splice donor site of intron 9 and a c.[1682C>T] mutation changing the threonine amino acid at position 561 to an isoleucine p.(T561I) (Fig. 2b). The splice mutant most likely induces aberrant splicing. However, it is not clear whether the end product is an aberrant protein or haploinsufficiency. Analysis of the parents for segregation indicated that the father was carrying the splice mutation while the mother was heterozygous for the p.(T561I) mutation (Fig. 2a). Neither mutation was seen in 96 ethnically matched controls. Analysis of the p.(T561I) mutation using Polyphen-2, a computer program that evaluates potential molecular mutations, generated a score of 1.00 out of a maximum of 1.00 indicating a probable damaging

C

Exon 8 : Mutation T561I

c.[2091+2T>C];[=]

p.[T561I];[=]

Exon 9 : Mutation IVS9+2T>C

c.[2091+2T>C] p.[T561I]

Fig. 2 a Pedigree of the family. Squares and circle denote male and female, respectively. LRP5 mutations are given under each symbol, parents are heterozygous, and child is compound heterozygous. b Electropherogram of the child showing the nucleotide sequences around

Homo sapien: Microcebus murinus mouse: Mus musculus: Rattus norvegius: Canis lupus familiaris: Felis Catus: Bos taurus: Equus caballus: Monodelphis domestica: Xenopus tropicalis: Tetraodon nigroviridis Takifugu rubripes: Danio reriro: Drosophila meganogaster:

FTLLGDFIYWTDWQRRSI **********T******* **********T******* **********T******* **********T******* **********T******* **********T******* **********T******* ******Y***T******* ******Y***T******* **********T***M*** **********T***M*** *****EY***T******* LSI*D*YL**T*******

the mutations in exon 8 and 9. Y is the code for C and T. c Alignment of the threonine (T) amino acid at position 561 of the LRP5 cDNA. Similar amino acids are represented by stars, different amino acids by their abbreviation

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mutation [15]. The alignment of T561 showed that it is very well conserved, even in Drosophila melanogaster and Danio rerio (Fig 2c). The T561I mutation was not observed by the Exome Aggregation Consortium (exac.broadinstitute.org) in more than 120,000 alleles.

References 1. 2.

3.

Discussion In normal eye development, hyaloid vessel regression during embryogenesis is dependent on ocular macrophages to induce apoptosis of endothelial cells [12]. This process fails to occur in Lrp5-deficient mice because the Wnt signaling that is activated in the physiologic elimination of fetal blood vessels is disrupted [5, 12, 13, 16]. In humans, PHPV can originate from mutations in NDP (familial exudative vitreoretinopathies, FEVR1), FRZ4 (FEVR2), or LPR5 (FEVR3). The patient described in this report presented bilateral vitreoretinal dysplasia distinct from FEVR but compatible with pseudoglioma. FEVR is a hereditary vitreoretinal disease that is initiated by the early arrest of peripheral retinal vascularization, leading to neovascularization, vascular leakage, retinal exudates, retinal folds, and retinal detachment in 20 % of affected subjects. The phenotype can vary from an asymptomatic state to a severe presentation resulting in blindness. FEVR is usually transmitted as an autosomal dominant (LRP5 and FZD4), autosomal recessive (LRP5), or X-linked recessive trait (NDP) [17]. LRP5 can thus cause dominant or recessive diseases affecting the eyes, bones, or both. Mutated LRP5 may induce a heterogeneous phenotype. In most cases, FEVR due to LRP5 mutations is associated with reduction of bone mineral density that starts during infancy [18], but cases without bone phenotype have been reported [19]. With more cases being diagnosed, it becomes clear that OPPG and FEVR are a continuum and should not be addressed as single entities. Accurate diagnosis of osteoporosis is important, and molecular analysis when available is highly recommended, in order to provide the best measures of prevention and/or treatment and facilitate genetic counseling.

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Acknowledgments We wish to thank this family for their help and patience, Ms. Angelique Schmid, Mr. Yann Leuba, and Mr. Cédric Schöpfer for their technical help, and Ms. Susan Houghton for editing the manuscript. Conflicts of interest None.

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19. Ethical approval The study was performed in adherence to the tenets of the Declaration of Helsinki and the parents authorized this publication.

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