Brittle Cornea Syndrome Associated with a Missense Mutation in the Zinc-Finger 469 Gene

Biochemistry and Molecular Biology Brittle Cornea Syndrome Associated with a Missense Mutation in the Zinc-Finger 469 Gene

Anne E. Christensen,1,2,3 Per M. Knappskog,1,3 Marit Midtbø,4 Clara G. Gjesdal,5 Jonas Mengel-From,6 Niels Morling,6 Eyvind Rødahl,2,3 and Helge Boman1,3

PURPOSE. To investigate the diverse clinical manifestations, for Biotechnology Information, Bethesda, MD) is a rare auto- identify the causative mutation and explain the association somal recessive disorder characterized by extreme thinning of with red hair in a family with brittle cornea syndrome (BCS). the cornea. Frequently, rupture of the cornea occurs as a result METHODS. Eight family members in three generations under- of minor trauma. Other ocular malformations include kerato- went ophthalmic, dental, and general medical examinations, conus, keratoglobus, and blue sclera. Systemic involvement is including radiologic examination of the spine. Bone mineral common, with manifestations such as joint hypermobility, skin density (BMD) and serum levels of vitamin D, parathyroid hyperelasticity, kyphoscoliosis, hearing defects, and dental ab- hormone, and biochemical markers for bone turnover were normalities. Hernias, syndactylia, and mental retardation have measured. Skin biopsies were examined by light and transmis- also been observed in patients with BCS. Red hair is associated with BCS in some families, although only 10 of 60 patients with sion electron microscopy. Molecular genetic studies included 1 homozygosity mapping with SNP markers, DNA sequencing, BCS have been reported to have red hair. Most individuals and MC1R genotyping. with BCS have been born to consanguineous parents and are thus expected also to be homozygous for a chromosomal RESULTS. At 42 and 48 years of age, respectively, both affected region adjacent to the BCS locus. Some of the nonocular individuals were blind due to retinal detachment and second- features in patients with BCS could therefore be due to ho- ary glaucoma. They had extremely thin and bulging corneas, mozygosity for mutations in closely linked genes. velvety skin, chestnut colored hair, scoliosis, reduced BMD, From the observed association of BCS with red hair, Zloto- dental anomalies, hearing loss, and minor cardiac defects. The gora et al.2 suggested that the BCS locus was closely linked to morphologies of the skin biopsies were normal except that in a gene responsible for hair color.2 Abu et al.3 assigned the BCS some areas slightly thinner collagen fibrils were seen in one of gene to a 4.7-Mb region on chromosome 16, region q24, close the affected individuals. Molecular genetic analysis revealed a 3 Ͼ to the melanocortin 1 receptor gene, MC1R. Recently, they novel missense mutation of ZNF469, c.10016G A, that was identified mutations in ZNF469, associated with BCS in Jewish predicted to affect the fourth of the five zinc finger domains of families of Tunisian origin and in a Palestinian family.4 The ZNF469 by changing the first cysteine to a tyrosine precise function of ZNF469 is not known. The protein has five (p.Cys3339Tyr). Both affected individuals were homozygous predicted zinc-finger domains in its C-terminal part and also for the common red hair variant R151C at the MC1R locus. shares approximately 30% homology with the helical parts of CONCLUSIONS. BCS is a disorder that affects a variety of connec- several collagens. Thus, both a regulatory and a structural role tive tissues. Reduced BMD and atypical dental crown morphol- in the assembly of collagen fibrils have been suggested.4 ogy have not been reported previously. The results confirm Most patients reported with BCS are from countries in the that BCS is associated with mutations in ZNF469. The associ- Middle East and North Africa. In 1968, a Norwegian family with ation with red hair in some individuals with BCS is likely to two children presenting with clinical features of BCS was occur by chance. (Invest Ophthalmol Vis Sci. 2010;51:47–52) described by Bertelsen5 and called dysgenesis mesodermalis DOI:10.1167/iovs.09-4251 corneae et sclerae (MIM 224200). In the present study, we reexamined this family. In this report, we describe the clinical rittle cornea syndrome (BCS; MIM 229200; Online Mende- and morphologic features of the affected individuals, and we Blian Inheritance in Man; http://www.ncbi.nlm.nih.gov/ show an association between the disorder in the family and a Omim/ provided in the public domain by the National Center missense mutation in the fourth zinc-finger domain of ZNF469. In contrast to their parents and sibs, both affected individuals had red hair. Genotyping of the melanocortin 1 receptor re- From the 1Center for Medical Genetics and Molecular Medicine vealed that the affected individuals were homozygous for a and the Departments of 2Ophthalmology and 5Rheumatology, Hauke- common variant of MC1R associated with red hair that coseg- 3 land University Hospital, Bergen, Norway; the Institute of Clinical regated with the ZNF469 mutation in this family. Medicine and the 4Department of Clinical Dentistry, Orthodontics and Facial Orthopedics, University of Bergen, Bergen, Norway; and the 6Section of Forensic Genetics, Department of Forensic Medicine, Uni- versity of Copenhagen, Copenhagen, Denmark. MATERIALS AND METHODS Supported by Grant 911296 from the Western Norway Regional Health Authority. Family Study Submitted for publication July 1, 2009; revised July 21, 2009; The family was first described in 1968, but was reexamined now as accepted July 21, 2009 part of the present study. Since 1968, no other affected family mem- Disclosure: A.E. Christensen, None; P.M. Knappskog, None; bers have been detected. Affected and unaffected family members M. Midtbø, None; C.G. Gjesdal, None; J. Mengel-From, None; N. Morling, None; E. Rødahl, None; H. Boman, None were invited to participate, and all signed an informed letter of con- Corresponding author: Eyvind Rødahl, Department of Ophthal- sent. The study was approved by the Regional Committees for Medical mology, Haukeland University Hospital, N-5021 Bergen, Norway; and Health Research Ethics, Western Norway (IRB 00001872), and [email protected]. adhered to the tenets of the Declaration of Helsinki. The parents, sibs,

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and offspring of the affected family members underwent a general medical examination with particular attention to skin, bone, and joints. Ophthalmic examination included slit lamp biomicroscopy, gonios- copy, Goldmann and Icare (Espoo, Finland) tonometry, corneal topog- raphy (Pentacam; Oculus Wetzlar, Germany), and axial length measurements (IOLMaster; Carl Zeiss Meditec, Jena, Germany; or CineScan, Quantel Medical, Clermont-Ferrand, France). Radiologic examination of the vertebral column and complete dental examinations including orthopantomograms were performed. Bone mineral density (BMD) was measured in the lumbar spine (L2–L4) and both hips by dual x-ray absorptiometry (Prodigy; GE Healthcare-Lunar, Madison, WI). Bio- chemical markers for bone turnover in serum, osteocalcin, bone-spe- ciﬁc alkaline phosphatase, and C-terminal cross-linking telopeptide of type I collagen were analyzed with kits from Immunodiagnostic Sys- tems Nordic (Herlev, Denmark).

Morphologic Studies Punch biopsies of upper arm skin were obtained from the affected individuals and from two age- and sex-matched healthy control sub- jects. For light microscopy, biopsies were fixed in 4% phosphate- buffered formaldehyde and embedded in paraffin, and 6-␮m sections FIGURE 1. Pedigree of the family with BCS. Filled symbols: affected were stained with hematoxylin and eosin. For transmission electron individuals. microscopy, biopsies were fixed in 2% glutaraldehyde in 0.2 M caco-

dylate buffer, embedded in Epon and postfixed in OsO4. Ultrathin sections were stained with 2% uranyl acetate and Reynold’s lead citrate she had a rupture of the left eye after falling on the floor. The before examination by transmission electron microscopy (JEM 1230; defect was sutured, and the cornea was covered with a Gun- JEOL, Tokyo, Japan). dersen conjunctival flap. Visual acuity was light perception, the corneal diameter was 14 mm, and there was excessive Homozygosity Mapping thinning of the cornea. At the age of 8, she again had a rupture of her left eye when it was struck with a finger while she was Genomic DNA was isolated from whole blood (QiaAmp kit; Qiagen, playing. The wound was closed, and she retained light percep- Hilden, Germany). A genome-wide single nucleotide polymorphism tion. She had varying eye pain, and was treated intermittently (SNP) scan was performed with a 50K chip (Affymetrix, Santa Clara, with antiglaucoma medication from the age of 8 to 14 years. CA) and a search for regions of homozygosity was performed with the 6 However, light perception was eventually lost. PLINK program. Renewed ocular examination at 48 years of age showed extensive scarring and band keratopathy (Fig. 2). In some DNA Sequencing and Mutation Detection areas, the cornea seemed to be without scars, but appeared PCR primers for amplification of exons and flanking intron sequences extremely thin, and the inner surface seemed to be coated with of ZNF469 were designed on computer (OLIGO software; National a thin iris-like structure. Intraocular pressure measured by Icare Bioscience, Plymouth, MN). DNA was amplified by PCR performed tonometry was 14 mm Hg. Ultrasound examination revealed an with standard procedures. After PCR amplification, the PCR products enlarged eye with no evidence of retinal detachment. were treated with SAP/exonuclease I (Amersham, Chalfont St. Giles, Patient IV-6 had blue sclerae at birth. He had a fracture of UK) and sequenced with dye termination chemistry (Prism BigDye the distal end of his right humerus after an injury a few days Terminator kit, ver 1.1; and a 3730 Genetic Analyzer; Applied Biosys- after birth. He was admitted for an eye examination when at tems, Inc., [ABI], Foster City, CA) and the DNA sequences analyzed the age of 1 month. He had a thin cornea with a diameter of 11 (SeqScape software; ABI). DNAs from 185 healthy local blood donors mm, and myopia of approximately Ϫ10 D. One year later, the were used as controls. corneal diameter was 12 mm. The corneal curvature was normal. At the age of 3 years, the corneal diameter was 13 mm, MC1R Genotyping and there was myopia of Ϫ23 D. Intraocular pressure was Thirteen sequence variants in MC1R of which 11 were associated with normal. At the age of 3.5 years, he had a rupture of the left eye red hair were examined by multiplex PCR single-base extension, and that was surgically closed. He then had two minor traumas to electrophoresis with multicolor fluorescence detection.7 his right eye at the age of 4 and 8 years, respectively, with rupture of Descemet’s membrane and subsequent development of corneal edema, which persisted from then on. At 9 RESULTS years of age, he suffered another rupture of the left eye, which again was sutured successfully, but visual acuity was gradually The family pedigree is shown in Figure 1. Two of five siblings lost. By the age of 10 years, the left eye showed no perception were affected. The parents were first cousins. In addition, of light. Corneal edema of the right eye varied from day to day, genealogical studies revealed that the parents also were more sometimes accompanied by a headache, and glaucoma was distantly related, as more than 20 common ancestral couples suspected. He was intermittently treated with topical timolol appeared in the pedigree 6 to 13 generations back (data not and pilocarpine. At the age of 28 years, he had a retinal shown). detachment of his right eye. Despite repeated surgery, the retina remained detached, and vision was subsequently lost. Ocular History Renewed ocular examination at 42 years of age revealed a Patient IV-4 was first seen at the age of 2 years when she bulging, thin cornea on both eyes (Fig. 2). The right cornea sustained a rupture of the right eye after a minor trauma. She was whitish, and the eye was filled with silicone oil which had blue sclerae and very thin corneae. Closure of the wound impaired intraocular examination. Intraocular pressure was was unsuccessful, and the eye was enucleated. One year later, between 15 and 21 mm Hg (Icare tonometry). The left cornea

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striae, and no signs of abnormal scarring. Joint mobility was within normal range. Radiologic examination of the spine showed scoliosis (Fig. 3). In IV-4 there was still a right/left double curve, a thoracic curve of 25° Cobb angle, apex at T8, and a thoracolumbar curve of 28° Cobb angle, apex at L2. In IV-6, the Cobb angle of the right thoracolumbar single curve scoliosis was 65°, apex at T9. The unaffected individuals III-1, III-2, IV-1, and IV-5 did not have any evidence of structural scoliosis based on radiologic examination. Clinical examination of individuals IV-2 and V-1 produced normal findings with respect to scoliosis. Involvement of different connective tissues is characteristic of BCS, and it was therefore of interest to examine BMD which had not been measured in patients with BCS previously. BMD of the hip and lumbar spine were in the range of osteopenia and osteoporosis, respectively for both of the affected individuals. Z-scores were lower in affected individuals than in unaffected family members, indicating a BMD lower than expected from age and sex (Table 1). Dental examination revealed that both of the affected siblings had normal occlusion (Angle class I), but with anterior crowding in the mandible. The incisors showed mineralization disturbances. Linear enamel hypoplasia was especially prominent on the mandibular canines and incisors of the affected woman (Fig. 4). The second premolars in the mandible dis- played atypical crown morphology with buccolingual com- pression and increased mesiodistal diameter (Fig. 4). The pan- oramic radiographs showed multiple pulp stones especially in molar teeth (data not shown). None of the healthy parents, siblings, or offspring had similar deviations. Cardiac examination of individual IV-4 revealed aortic valve insufficiency grade I-II, and a slight mitral valve insufficiency. Individual IV-6 had been diagnosed with supraventricular

FIGURE 2. Slit lamp photograph of the eyes of individual IV-4 (A, left eye) and IV-6 (B, right eye; C, left eye).

was scarred in its central part and seemed to be lined with a pigment layer on the internal surface, probably an atrophic iris. Ultrasound B-scan showed retinal detachment in both eyes. The axial length was 26.7 and 22.5 mm of the right and left eye, respectively. Unaffected family members underwent a thorough ocular examination. Visual acuity, intraocular pressure (Goldmann tonometry), axial length, corneal diameter, and central corneal thickness were all within the normal range. Structurally, the eyes also appeared normal. Nonocular Features From childhood, both affected individuals were noted to have chestnut hair, as opposed to their siblings. They also had a more slender stature. Radiologic examination of the vertebral column was ﬁrst performed at the age of 14 (IV-4) and 8 years (IV-6), respectively. Both had normal bone structure. In IV-4 there was a slight scoliosis with a right/left double curve, a thoracic curve of 9° Cobb angle, apex at T8, and a thoracolumbar curve of 10° Cobb angle, apex at L2. No scoliosis was seen in IV-6 at this age, but it manifested in his early teens, and FIGURE 3. Radiologic examination of the vertebral column of affected he was then wearing a brace for 18 months (data not shown). individuals IV-4 (A) and IV-6 (B) and sibling IV-5 (C) showing scoliosis The present examination revealed that the family members with right/left double curve in IV-4 and a right thoracolumbar single had velvety skin, but with normal elasticity, no pathologic curve in IV-6.

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TABLE 1. BMD Measurements

Z-Score Total Hip Individual Z-Score Spine (Sex, Age) Right Left L2–L4

III-1 (M 84 y) Ϫ0.5 Ϫ0.8 ϩ1.1 III-2 (F 79 y) 0.0 Ϫ0.2 Ϫ1.2 IV-1 (F 53 y) Ϫ0.2 Ϫ0.6 Ϫ0.8 IV-2 (F 51 y) Ϫ0.4 Ϫ0.7 0.6 IV-4 (F 48 y) ؊0.8 ؊1.0 ؊2.3 IV-5 (M 47 y) ϩ1.0 ϩ0.9 ϩ3.4 IV-6 (M 42 y) ؊2.4 ؊2.2 ؊2.6 V-1 (M 20 y) ϩ1.3 ϩ1.4 ϩ0.8 FIGURE 5. Mutation analysis of the ZNF469. Partial sequence chro- matograms showing the wild-type (WT) DNA sequence of a normal Z-scores represent standard deviations from age- and sex-speciﬁc person and the DNA sequence of an affected individual homozygous mean value. The data for the individuals reported in this study are in for the ZNF469 c.10016 GϾA mutation. bold.

tachycardia and had been treated with metoprolol. Cardiac Biochemical Analysis ultrasonography showed a mitral valve insufficiency grade II, Parathyroid hormone (PTH) and 25-OH vitamin D were normal and a tricuspid valve insufficiency grade I. None of the valve in all individuals except for IV-6 who had a mild vitamin D insufficiencies were of functional importance. deficiency (23 nM; normal range 50 to 113 nM), with slightly Both affected individuals also presented with a hearing increased PTH (9.3 pM; normal range 1.3–6.8 pM). Biochemi- defect. IV-4 had a small conductive hearing loss with a small cal markers of bone turnover, osteocalcin, bone-specific alka- dip at 1000 Hz and a hearing level at 30 dB. IV-6 had a line phosphatase and C-terminal cross-linking telopeptide of combined hearing loss with hearing level 40 and 65 dB for the type I collagen were within the normal range in all individuals. left and right ear, respectively, and no hearing in the higher frequencies above 4000 Hz. The hearing had deteriorated over Morphologic Studies the years due to a sensorineural hearing loss. IV-6 also had additional problems with vertigo of probable inner ear origin. Light microscopy findings in the cornea from the enucleated eye of IV-4 have been published in detail.5 Light microscopy of skin biopsies did not reveal any gross abnormalities of the architecture of the skin. We did not observe the “holes” in the dermis reported by Royce et al.8 Transmission electron microscopy revealed collagen fibrils in IV-4 to have a normal diameter. In IV-6, we observed minor variation in the diameter of the fibrils, and slightly thinner fibrils could be seen in some areas (data not shown). Genetic Analysis Genome-wide testing with SNP markers identified two large candidate regions of homozygosity on chromosomes 9 and 10 and a 5.8-Mb (15 cM) region distal to the SNP marker rs962878 at the terminal end of the long arm of chromosome 16. This region encompassed 114 established and predicted genes, including ZNF469 and MC1R. DNA sequencing of ZNF469 revealed a missense mutation c.10016GϾA (Fig. 5). This mutation was not seen in a panel of 185 blood donors (370 chromosomes). The mutation is predicted to alter the first cysteine in the fourth zinc-finger domain of the protein to a tyrosine (p.Cys3339Tyr). To identify the genetic basis of red hair in this family, 13 sequence variants in MC1R of which 11 were associated with red hair were analyzed. Both affected individuals were homozygous, and both parents and one of the sibs were heterozygous for the most common red hair variant in the Caucasian population, R151C.7

DISCUSSION In the present report we have identiﬁed a novel mutation in ZNF469 in the family originally described with “mesodermal dysgenesis of the cornea and sclera” (MIM 224200). The clinical presentation and the morphologic features as well as the FIGURE 4. Photographs of the teeth of affected individuals IV-4 (A, C) and IV-6 (B). Linear hypoplasias can be deﬁned on the teeth of indi- observed mutation show that the affected family members vidual IV-4 (A), along with atypical crown morphology of the right have the condition now known as BCS (MIM 229200). Het- (inset a) and left (inset b) second mandibular premolars (arrows), with erozygous individuals appear to be indistinguishable from compressed buccolingual and increased mesiodistal diameter. healthy individuals.

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Rupture of the cornea either spontaneously or due to minor Dental anomalies (dentinogenesis imperfecta) have been trauma is the most dramatic feature of BCS. In 31 of 60 patients described previously in 10 of 60 patients with BCS.1 The with BCS, rupture of the cornea has been reported in 44 affected individuals examined in the present study both had instances.1 The left eyes of IV-4 and IV-6 were surgically re- dentinogenesis imperfecta-like mineralization disturbances, paired after corneal rupture, but then visual function was but few other characteristics. In human teeth, there is a broad gradually lost. At present, the atrophic iris appears to be at- spectrum of inherited dentin malformations with unknown tached to the posterior surface of the cornea, and impaired etiology.14 The association of a dentin malformation with a drainage from the anterior chamber may have occurred. Raised mutation in the ZNF469 may therefore add to our understand- intraocular pressure has not been observed, but interpretation ing of these conditions. Atypical crown morphology is a com- has been difficult due to scarring and the reduced rigidity of mon observation in many genetic disorders. In our patients, the extremely thin cornea. Secondary glaucoma after rupture the lower second premolars are deviant, indicating that the repair has been reported in other patients with BCS9 and is the defective gene is expressed in the series of epithelial–mesen- 15 most likely cause of visual loss in the injured eyes of our chymal interactions before mineralization. patients. Development of corneal edema in patients with BCS The cardiac abnormalities observed are common in healthy 16 has been observed as a consequence of detachment10 or individuals. Valve deficiencies have been noted in other pa- 9 tears11 of Descemet’s membrane, which is most likely the tients with BCS including pulmonary stenosis and mitral valve 11 cause of corneal edema in the right eye of IV-6. Many patients prolapse. In contrast to EDS type VI where arterial ruptures with BCS are myopic. The right eye of IV-6 was myopic (Ϫ23 are common, patients with BCS appear to be less susceptible to D) and had an axial length of 26.7 mm. The myopia cannot be cardiovascular problems. Hearing defects have been observed in 20 of 60 patients explained only by the increase in axial length. Keratoglobus 17 with thinning of the cornea and altered corneal curvature are with BCS. Conductive hearing loss, as seen in IV-4 ; sensorineural hearing loss2,10; and combined hearing loss (individual also likely contributors. Patient IV-6 had retinal detachment 12 that eventually led to blindness in his uninjured, right eye. IV-6) have been reported. The basis for the combined hear- Retinal detachment has rarely been reported in patients with ing loss is uncertain. A CT scan of IV-6 did not show otoscle- BCS.12 Most patients, however, have been examined at a young rotic foci. age, and retinal detachments may therefore not have occurred The morphology of the skin biopsies was virtually identical yet. with that of healthy controls, both at the light and electron Involvement of connective tissues in organs other than the microscopic level, except for minor variation in the diameter corneae is common in BCS, including skin hyperelasticity, joint of collagen fibrils in one of the affected individuals. Several hypermobility, kyphoscoliosis, and dental anomalies.1 Clini- investigators have examined skin biopsies from patients with BCS. In one report, 40- to 60-␮m holes were described in the cally, the differential diagnosis of BCS and Ehlers-Danlos syn- dermis.8 Other investigators have observed minor variation in drome (EDS) type VI can be difficult. Type VI is the kyphosco- collagen fibril diameter,18 as seen in IV-6. In general, however, liotic form of EDS (MIM 225400) and is associated with the morphology of the skin has been rather unremarkable with mutations in the lysyl-1-hydroxylase gene. In general, the ocu- respect to both the gross architecture and the structure of the lar features are more prominent and the systemic manifesta- collagen fibrils. tions less severe in BCS than in EDS type VI.13 BCS was once Testing for homozygosity revealed a candidate region that thought to be a subtype of EDS (type VIB), but after the encompassed the chromosome 16q24 region identified by Abu identification of causative mutations in ZNF469, BCS must et al.3 They sequenced the genes in this region and identified clearly be considered a separate entity. Ͼ two frameshift mutations in the ZNF469 that were associated BMD values 2 SD below age- and sex-specific mean values with the disease.4 By sequencing the entire ZNF469,weob- were found in the lumbar spine of the two affected persons. served homozygosity for a missense mutation that was pre- This is expected in less than 2% of the normal population. dicted to affect the fourth of the five zinc finger domains of Scoliosis was mild in this part of the vertebral column and is ZNF469 by changing the first cysteine to a tyrosine. This not likely to have affected the results. Individual IV-6 was cysteine is conserved in many species, including chimpanzee, slightly deficient in vitamin D, and this could partly explain his dog, mouse, and rat, suggesting that the missense mutation low BMD. Sedentary lifestyle may predispose for bone loss and could alter a functionally important part of the protein. Our reduced peak BMD in adults, but it is not likely to be the cause observation thus confirms that mutations in ZNF469 are the of reduced BMD in the affected individuals, since they have likely cause of BCS. had regular physical activity, both as children and adults. Re- The first clues to the localization of a genetic locus for BCS duced BMD measurements have not been reported previously was offered when red hair was associated with BCS in Tunisian in BCS, but BMD must be determined in additional patients Jews.2 Red hair is a highly unusual trait in this population. As before the conclusion can be reached that reduced BMD is part one affected person did not have red hair, it was assumed that of BCS. red hair was not a manifestation of a mutant BCS gene, but High levels of biochemical markers for bone turnover, indi- rather that the loci for the two traits were closely linked and a cating increased remodeling of the trabecular bone, are usually crossover had occurred. Red hair is not an uncommon trait in seen in patients with osteoporosis due to bone loss. There the Norwegian population. The sum of the highly penetrant were no indications of increased bone turnover in the affected MC1R allele variants for red hair R142H, R151C, R160W, participants of our study. Whether the low BMD observed in D294H, and D84E in Caucasian populations is ϳ 28%.7 Thus, BCS patients is due to structural changes in bone or increased the chance that a BCS mutation would occur on a chromosome bone loss remains to be determined. with such a variant is correspondingly high. Since the affected Pathologic fractures, a typical feature of osteogenesis im- sibs in this family are homozygous for the BCS-MC1R chromo- perfecta, is usually not seen in BCS. Although IV-6 had a somal region, the chance of observing red hair in BCS is also fracture of his right humerus a few days after birth, he has not ϳ28%. Thus, it is likely that the association of BCS and red hair had any fractures since. His affected sister IV-4 has never occurs by chance. Most of the other features seen in BCS are experienced any fracture. Thus, pathologic fractures do not likely to be the result of connective tissue defects, and may seem to occur in the affected individuals, despite the presence represent a pleiotropic effect of the ZNF469 mutations. We of reduced BMD. cannot exclude, however, that variants in other syntenic genes

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cosegregating with ZNF469 may play a role in the develop- 8. Royce PM, Steinmann B, Vogel A, et al. Brittle cornea syndrome: an ment of the various facets of brittle cornea syndrome. heritable connective tissue disorder distinct from Ehlers-Danlos syndrome type VI and fragilitas oculi, with spontaneous perfora- Acknowledgments tions of the eye, blue sclerae, red hair, and normal collagen lysyl hydroxylation. Eur J Pediatr. 1990;149:465–469. The authors thank Jorunn S. Bringsli, Sigrid Erdal, Guri Matre, Unni 9. Behrens-Baumann W, Gebauer HJ, Langenbeck U. Syndrome of Larsen, and Bård Kjersem for skillful technical assistance, and Johan G. blue sclerae and keratoglobus (ocular type of Ehlers-Danlos syn- Brun, Lisbeth Sviland, Per Møller, Per T. Nilsen, Anne Kask, Jostein drome (in German). Albrecht Von Graefes Arch Klin Exp Ophthal- Kråkenes, and Bjørg Almås for professional assistance. The transmis- mol. 1977;204:235–246. sion electron microscopy was performed at the Molecular Imaging 10. Judisch GF, Waziri M, Krachmer JH. Ocular Ehlers-Danlos syn- Center (FUGE, Norwegian Research Council), University of Bergen. drome with normal lysyl hydroxylase activity. Arch Ophthalmol. 1976;94:1489–1491. References 11. Cameron JA, Cotter JB, Risco JM, Alvarez H. Epikeratoplasty for keratoglobus associated with blue sclera. Ophthalmology. 1991; 1. Al-Hussain H, Zeisberger SM, Huber PR, et al. Brittle cornea syn- 98:446–452. drome and its delineation from the kyphoscoliotic type of Ehlers- 12. Izquierdo L Jr, Mannis MJ, Marsh PB, et al. Bilateral spontaneous Danlos syndrome (EDS VI): report on 23 patients and review of the corneal rupture in brittle cornea syndrome: a case report. Cornea. literature. Am J Med Genet A. 2004;124:28–34. 1999;18:621–624. 2. Zlotogora J, BenEzra D, Cohen T, Cohen E. Syndrome of brittle 13. Beighton P, De Paepe A, Steinmann B, et al. Ehlers-Danlos cornea, blue sclera, and joint hyperextensibility. Am J Med Genet. syndromes: revised nosology, Villefranche. 1997. Ehlers-Danlos 1990;36:269–272. National Foundation (USA) and Ehlers-Danlos Support Group 3. Abu A, Frydman M, Marek D, et al. Mapping of a gene causing (UK). Am J Med Genet. 1998;77:31–37. brittle cornea syndrome in Tunisian Jews to 16q24. Invest Oph- 14. Kim JW, Simmer JP. Hereditary dentin defects. J Dent Res. 2007; thalmol Vis Sci. 2006;47:5283–5287. 86:392–399. 4. Abu A, Frydman M, Marek D, et al. Deleterious mutations in the 15. Hu JC, Simmer JP. Developmental biology and genetics of dental zinc-finger 469 gene cause brittle cornea syndrome. Am J Hum Genet. 2008;82:1217–1222. malformations. Orthod Craniofac Res. 2007;10:45–52. 5. Bertelsen TI. Dysgenesis mesodermalis corneae et sclerae. Rupture 16. Bonow RO, Lakatos E, Maron BJ, Epstein SE. Serial long-term of both corneae in a patient with blue sclerae. Acta Ophthalmol assessment of the natural history of asymptomatic patients with (Copenh). 1968;46:486–491. chronic aortic regurgitation and normal left ventricular systolic 6. Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for function. Circulation. 1991;84:1625–1635. whole-genome association and population-based linkage analyses. 17. Greenfield G, Stein R, Romano A, Goodman RM. Blue sclerae and Am J Hum Genet. 2007;81:559–575. keratoconus: key features of a distinct heritable disorder of con- 7. Mengel-From J, Borsting C, Sanchez JJ, et al. Determination of nective tissue. Clin Genet. 1973;4:8–16. cis/trans phase of variations in the MC1R gene with allele-specific 18. Ogur G, Baykan N, De Paepe A, et al. Clinical, ultrastructural and PCR and single base extension. Electrophoresis. 2008;29:4780– biochemical studies in two sibs with Ehlers-Danlos syndrome type 4787. VI-B-like features. Clin Genet. 1994;46:417–422.

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