ß 2007 Wiley-Liss, Inc. American Journal of Medical Part A 143A:1790–1795 (2007)

Research Letter Abnormalities in Two Patients With Features of Autosomal Dominant Robinow Syndrome

Juliana F. Mazzeu,1 Ana Cristina Krepischi-Santos,1 Carla Rosenberg,1 Karoly Szuhai,2 Jeroen Knijnenburg,2 Janneke M.M. Weiss,3 Irina Kerkis,1 Zan Mustacchi,4 Guilherme Colin,5 Roˆmulo Mombach,6 Rita de Ca´ssia M. Pavanello,1 Paulo A. Otto,1 and Angela M. Vianna-Morgante1* 1Centro de Estudos do Genoma Humano, Departamento de ´tica e Biologia Evolutiva, Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil 2Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands 3Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands 4Hospital Infantil Darcy Vargas, Sa˜o Paulo, Brazil 5Departamento de Gene´tica Me´dica, Univille, Joinville, Brazil 6Centrinho Prefeito Luiz Gomes, Secretaria Municipal de Sau´de, Joinville, Brazil Received 13 April 2006; Accepted 13 December 2006

How to cite this article: Mazzeu JF, Krepischi-Santos AC, Rosenberg C, Szuhai K, Knijnenburg J, Weiss JMM, Kerkis I, Mustacchi Z, Colin G, Mombach R, Pavanello RM, Otto PA, Vianna-Morgante AM. 2007. Chromosome abnormalities in two patients with features of autosomal dominant Robinow syndrome. Am J Med Genet Part A 143A:1790–1795.

To the Editor: Patient 1 Robinow syndrome [OMIM 180700] is characteriz- At age 3 4/12 years the girl was diagnosed as ed by fetal facies, mesomelic dwarfism, and hypo- affected by DRS (Fig. 1A). Detailed clinical examina- plastic genitalia. An autosomal recessive (RRS) and tion at age 9 4/12 years showed (117 cm; an autosomal dominant form (DRS) of the syndrome <3rd centile), frontal bossing, hypertelorism (ICD: have been described, the former presenting with 4.0 cm > 97th centile, OCD: 11.5 cm > 97th centile), more severe skeletal anomalies. The gene mutated in down-slanted palpebral fissures, facial nevus, stra- RRS has been identified as ROR2 at 9q22 [Afzal et al., bismus, short nose with mildly anteverted nares, 2000; Van Bokhoven et al., 2000], and encodes a depressed nasal bridge, long philtrum, microretrog- tyrosine–kinase receptor involved in cell growth and nathia, large downturned mouth with thin upper lips, differentiation. The gene associated with DRS has not highly arched palate, and posteriorly rotated . been identified. We describe two unrelated patients Her teeth were hypoplastic and malaligned. She had with clinical pictures of DRS and chromosome pectus excavatum. Both hands showed a single 1 abnormalities. In one patient, a duplication of palmar crease and fifth finger clinodactyly. Webbing chromosome 1p [46,XX,dir dup(1)(p13p31)] was of the first and second toes was present bilaterally. identified by G-banding, and the breakpoints were Radiographs showed rhizomesomelic dysplasia; no mapped by fluorescent in situ hybridization (FISH). vertebral anomalies were observed. Examination of In the other patient, array–CGH analysis revealed a the genitalia showed hypoplastic and microdeletion of chromosome 1q (1q41 ! q42.1). The finding of different chromosome rearrange- ments in DRS patients points to genetic hetero- geneity in the phenotype, and mapped to rearranged segments appear as candidates for the Grant sponsor: FAPESP, CAPES, CNPq. syndrome. *Correspondence to: Angela M. Vianna-Morgante, Departamento de This study has been approved by the institutional Gene´tica e Biologia Evolutiva, Instituto de Biocieˆncias, Universidade de Sa˜o Paulo; C.P.11461; 05422-970-Sa˜o Paulo, SP, Brazil. ethics committee, and the families provided written E-mail: [email protected] informed consent. DOI 10.1002/ajmg.a.31661 American Journal of Part A: DOI 10.1002/ajmg.a

ROBINOW SYNDROME 1791 showing that the maternally inherited chromosome had the duplicated segment (data not shown). Both parents had normal . Probes cloned in YACs (CEPH, Paris) and BACs (CHORI, Oakland) mapping to the short arm of (The GDB Database (GDB), www.gdb.org; and National Center for Biotechnol- ogy Information (NCBI), www.ncbi.nih.gov) were used to delimit the duplicated segment and map the breakpoints by FISH (Fig. 2A). Conditions for probe hybridization and detection were as pre- viously described [Rosenberg et al., 1994]. Double- FIG.1. A: Patient 1 at age 3 4/12 years; B: Patient 2 at age 6 months. color hybridization confirmed that the duplication was direct (Fig. 2D). BAC RP11-585M16 contained the distal breakpoint at 1p31.1 (Fig. 2C), and the minora, but normal sized . The patient was partially overlapping BACs RP11-155D24 and RP11- developmentally delayed and could not speak. She 140L8 contained the proximal breakpoint at 1p13.3 sat at 7 months of age and walked at 4 8/12 years. (Fig. 2B). Audiometry was normal. There were recurrent To determine the parental origin of the duplicated infections. Bone length measures on radiographs segment, genotyping of six microssatelite loci at age 9 4/12 years confirmed the presence of (D1S2792, D1S2778, D1S248, D1S1623, D1S1163, rhizomesomelic upper limb shortening, as revealed D1S406) mapped to the duplicated segment was by ratios radius/humerus (0.72 at left, 0.71 at right; performed by standard radioactive (32P) PCR, using 5th centile, indicating preponderant mesomelic the patient’s and their parent’s genomic DNA shortening), radius/tibia (0.58 at both sides; <5th extracted from peripheral blood lymphocytes. centile, indicating upper limb shortening), and tibia/ Primer sequences were obtained from GDB. The femur (0.90 at left, 0.84 at right; 50th–95th centile, patient was heterozygous for four loci mapped indicating no marked length difference between the within the duplicated segment (D1S2778, D1S248, two lower limb segments). D1S1623, D1S1163), but three different alleles were Chromosome analysis after G-banding, in peri- not identified in any of these loci. We performed pheral blood lymphocytes, showed a direct inter- dosage analysis for loci D1S1623, D1S1163 based on stitial duplication of the short arm of chromosome the ratios of band optical densities (OD) in auto- 1,46,XX,dir dup(1)(p13p31). C-banding showed an radiograms [Antonini et al., 2002] in the patient and increased polymorphic pericentromeric heterochro- her mother. The maternally inherited alleles were the matic region on the duplicated chromosome 1. This more amplified ones, thus pointing to the maternal variant was also present in the patient’s mother origin of the duplicated segment (data not shown).

FIG.2. Patient 1—Breakpoint mapping of 1p duplication by FISH: A: YAC (left) and BAC (right) clones hybridized. White boxes: nonduplicated clones; black boxes: duplicated clones; gray boxes: breakpoint-containing clones; B: At the proximal breakpoint, BAC RP11-155D24 mapped to 1p13.3 was duplicated, but the distal signal was less intense; C: at the distal breakpoint, BAC RP11-585M16 mapped to 1p31.1, also duplicated, produced a diminished proximal signal. D: Demonstration of the direct nature of the duplication by double-color hybridization of YACs 881f6 (green) and 963f5 (red). American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

1792 MAZZEU ET AL. Patient 2 We compared the clinical findings between Patient 1 and 4 previously described carriers of similar 1p At birth the boy’s length was 48 cm (< 10th centile) duplications [Mohammed et al., 1989; Hoechstetter and weight, 3780 g (75th centile). At clinical examina- et al., 1995; Dhellemmes et al., 1998; Garcia-Heras tion at age 2 years he presented with delayed et al., 1999]. The localization of breakpoints was neuropsychomotor development, generalized hypo- based on G-banding, and therefore different break- tonia, and hemiparesis at right. He had short stature points might be involved in these rearrangements. (84 cm, 3rd centile), coarse facies, midface hypopla- None of the literature cases had a clinical picture sia, hypertelorism (ICD: 3.1 mm; OCD: 9.4 mm both > suggestive of DRS, although some DRS signs were 98th centile), upslanted palpebral fissures, long eye- present in most of them, such as hypertelorism (DRS- lashes, blue sclerae, convergent strabismus, wide flat 100%), (DRS-81%), clinodactyly (DRS- nasal bridge, bulbous nose with anteverted nares, 70%), and micrognathia (DRS-56.7%). The only male long well marked philtrum, triangular mouth, down- patient had cryptorchidism (DRS-71.6%), and one slanted mouth corners, thin upper lip, cleft palate, patient [Dhellemmes et al., 1998] had rhizomelic gum hyperplasia, micrognathia, thick ear lobes with shortening (DRS-35.4%), also present in our patient hyperfolded helix, and short neck (Fig. 1B). Examina- [frequencies in DRS according to Mazzeu et al. tion of the genitalia showed and right (2007)]. cryptorchidism. Upper arms presented with mesome- All previous reports of deletions of chromosome 1q lic shortening with limited elbow supination. Hands encompassing 1q41 and/or 1q42 were based on G- showed metacarpal shortening, wide thumbs, and banding analysis without precise mapping of break- nail dysplasia. He had congenital club foot, large first points, and are much larger than that described in toes, and widely spaced first and second toes. He had Patient 2; therefore, their precise overlapping with skin laxity and well marked palmar and plantar the deletion in our patient could not be established creases. Ophthalmologic examination revealed [Andrle et al., 1978; Kessel et al., 1978; Molina et al., hypermetropia, glaucoma, nystagmus, and alternat- 1978; Dignan and Soukup, 1979; Neu et al., 1982; ing strabismus. An ECG performed at 9 months of age Turleau et al., 1983; Beemer et al., 1985; Johnson revealed reflux of tricuspid valve and thickening of et al., 1985; Al-Awadi et al., 1986; Tolkendorf et al., pulmonary valve. X-rays documented enlarged 1989]. The most common signs observed in the femoral epiphysis, a higher distance between femoral described patients were low birth weight, delayed heads and iliac bones, and kyphosis. Brain MRI neuropsychomotor development/ mental retarda- showed left-brain hemiatrophy with cortical dyspla- tion, short stature, microcephaly, slanted palpebral sia, and possible frontal microgyria at right. fissures, epicanthal folds, hypertelorism, short and The presence of glaucoma (that may represent a wide nose, long and smooth philtrum, downturned manifestation of an anterior eye-chamber anomaly), mouth corners, and microretrognatia. Heart pro- short stature, and developmental delay raised the blems were observed in 4/11 cases. Our patient did possibility of the child having the Peters Plus not present with low birth weight, microcephaly, syndrome, which has recently been associated with epicanthal folds, or smooth philtrum. However, he mutations in the B3GALTL gene [Lesnik et al., 2006]. had gum hyperplasia, mesomelic limb shortening, The sequencing of exon 8, the described hotspot nail dysplasia, clubfoot, hypermetropia, brain hemi- for the causative mutations, and exon 5, in which atrophy, and cortical dysplasia, clinical signs not a pathogenic mutation has also been identified, described previously in association with chromo- revealed no pathogenic mutations. some 1q41 and/or 1q42 deletions. The patient No chromosome abnormalities were identified reported by Johnson et al. [1985] had skin laxity after G-banding. Array–CGH analysis was per- and deep palmar and plantar creases also present in formed using DNA from peripheral blood lympho- our patient. cytes, as previously described Rosenberg et al. [2006]. It is noteworthy that both patients here described The slides containing triplicates of 3,500 large present with clinical signs frequent in autosomal DRS insert clones spaced at 1.0 Mb density over the full (Table I). Patient 1 presents with 18 out of 21 genome were produced at the Leiden University pertinent signs present in more than 50% of DRS Medical Center. Information regarding the full set of patients, and Patient 2, 19 out of 22 signs. These clones is available at the Wellcome Trust Sanger findings are suggestive of candidate regions for the Institute mapping database site, Ensembl [http:// syndrome on chromosome 1. www.ensembl.org/]. Probes RP11-308113 (1q41), Chromosome abnormalities have been previously RP11-239e10 (1q41-q42.1), and RP11-105I12 described in two girls with the diagnosis of Robinow (1q42.1) were found to be deleted (Fig. 3A). We syndrome. In one girl, a deletion of chromosome 7, confirmed the deletion by FISH of corresponding del(7)(q32qter), was detected [Wang et al., 1997]. probes (Fig. 3B) to patient metaphases. Neither The other patient carried a chromosome 1p deletion, parent carried the deletion as shown by FISH (data del(1)(p22p32) [Sivasankaran et al., 1997]. Clinical not shown). signs of Robinow syndrome are not typically found in American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

ROBINOW SYNDROME 1793

FIG.3. Patient 2—A: Array–CGH profile of chromosome 1 shows deletion of clones RP11-308I13, RP11-239E10, and RP11-105I12 (red spots); the position of the deletion is depicted on chromosome 1 idiogram. Underneath, enlargement of the deleted segment, including clones and genes. B: FISH of clone RP11-239E10 confirming the chromosome 1 deletion (arrow). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] patients with similar 1p [Mircher et al., 2003] or 7q duplicated segment in our patient. VAV3 belongs to a deletions [Verma et al., 1992]. The 1p duplication family of GEFs for Rho GTPases, acting on the actin here described and the deletion present in the RS cytoskeleton rearrangement [Hornstein et al., 2004]. patient of Sivasankaran et al. [1997] include common It is a functionally similar to FGD1, and segments, thus pointing to a candidate region for FGD1 gene mutations cause Aarskog syndrome, a the syndrome on the short arm of chromosome 1. A condition with several clinical signs in common with gene damaged by the duplication breakpoint could RS. A third candidate gene on 1p is ROR1, which be deleted in the other case. Alternatively, over- codes for a member of the ROR family of tyrosine– expression could occur due to gene duplication or kinase receptors, sharing 58% identity with ROR2 otherwise, to the deregulation of genes outside the protein. Mutations in ROR2 cause recessive Robinow deleted segment. Approximately 140 known genes syndrome [Afzal et al., 2000; Van Bokhoven et al. map within the 34 Mb duplicated segment in 2000]. ROR1 maps to 1p31.3 and, although deleted in Patient 1 (NCBI). We looked for candidate genes for the patient of Sivasankaran et al. [1997], it is not DRS on 1p based on their location, function, and duplicated in our patient, mapping approximately similarity to genes known to be mutated in syn- 10 Mb from the distal breakpoint. A positional effect dromes sharing clinical signs with RS. BAC RP11- or the disruption of a long-distance regulatory region 585M16, which contains the distal breakpoint, spans should be considered in this case [Fritz et al., 2005; gene TNNI3K. Although this gene may be disrupted Milot et al., 1996]. Studies in mice show that Ror1 and by the duplication breakpoint, TNNI3K is a kinase Ror2 associate to different cytoskeletal structures, expressed specifically in cardiac myocytes [Zhao Ror1 co-localizing with F actin and Ror2 with et al., 2003], and therefore does not appear as a microtubules [Paganoni et al., 2004]. Therefore, it is candidate for DRS. The proximal breakpoint, con- reasonable to consider that dominant Robinow tained in partially overlapped BACs RP11-140L8 and syndrome may be caused by mutations in genes, RP11-155D24, do not span any known gene. Another like ROR1 and VAV3, both involved in cytoskeletal candidate gene is VAV3 mapped at 1p13, within the arrangement. American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

1794 MAZZEU ET AL.

TABLE I. Clinical Signs Present in More than 50% of the Patients brachydactyly type B, is caused by mutation of ROR2. Nat With Autosomal Dominant Robinow Syndrome (DRS)* and Their Genet 25:419–422. Presence in the Patients Herein Described Al-Awadi SA, Farag TI, Usha R, El-Khalifa MY, Sundareshan TS, Al-Othman AS. 1986. Brief clinical report: Interstitial deletion Frequency of the long arm of chromosome 1 (del(1)(q32q42)). Am J Med Clinical signs in DRS (%)* Patient 1 Patient 2 Genet 23:931–933. Andrle M, Erlach A, Mayr WR, Rett A. 1978. Terminal deletion of Hypertelorism 100 þþ (1)(q42) and its phenotypical manifestations. Hum Genet 41: Wide nasal bridge 100 þþ 115–120. Anteverted nares 100 þþ Antonini S, Kim CA, Sugayama SM, Vianna-Morgante AM. 2002. Upturned nose 86.7 þþ Delimitation of duplicated segments and identification of their Micropenis 84.1 Female þ parental origin in two partial chromosome 3p duplications. Short stature 81.2 þþ Am J Med Genet 113:144–150. Short nose 81.2 þþ Beemer FA, Klep-de Pater JM, Sepers GJ, Janssen B. 1985. Two Brachydactyly 81 cases of interstitial deletion of the long arm of chromosome 1 Midface hypoplasia 80.6 þþ del(1)(q21 q25) and del(1)(q41 q43). Clin Genet 27: Mesomelic limb shortening 80.1 þþ ! ! 515–519. Prominent forehead 79 þ Dhellemmes C, Choiset A, Narbouton R, Girard S, Tapia S, Thepot Depressed nasal bridge 77.9 þþ F, Sarrut S. 1998. Interstitial dup (1p) and severe intrauterine Cryptorchidism 71.6 Female þ growth retardation. Ann Ge´ne´t 2:129–131. Clinodactyly 70 þ Dignan PS, Soukup S. 1979. Terminal long-arm deletion of Triangular mouth 64.9 þþ chromosome 1 in a male infant. Hum Genet 48:151–156. Long philtrum 64.7 þþ Fritz B, Kunz J, Knudsen GP, Louwen F, Kennerknecht I, Eiben B, 64.2 Orstavik KH, Friedrich U, Rehder H. 2005. Situs ambiguus in a Down-slanted mouth corners 62.9 þþ female fetus with balanced (X;21) translocation—evidence for Short hands 61.5 þ functional nullisomy of the ZIC3 gene? Eur J Hum Genet 13: Micrognathia 56.7 þþ 34–40. Long eyelashes 54 þþ Garcia-Heras J, Corley N, Garcia MF, Kukolich MK, Smith KG, Day Highly arched palate 51.5 þþ DW. 1999. De novo partial duplications 1p: Report of two Hypoplastic labia minora 50.4 þ Male cases and review. Am J Med Genet 82:261–264. *Mazzeu et al. [2007]; (þ) present; () absent. Hoechstetter LH, Soukup S, Schorry EK. 1995. Familial partial duplication (1)(p21p31). Am J Med Genet 59:291–294. Hornstein I, Alcover A, Katzav S. 2004. Vav , masters of the world of cytoskeleton organization. Cell Signal 16:1–11. On the other hand, Patient 2 presented with a Johnson VP, Heck LJ, Carter GA, Flom JO. 1985. Deletion of the deletion of chromosome 1q. Eight known genes, distal long arm of chromosome 1: A definable syndrome. Am J Med Genet 22:685–694. which play essential roles during development, map Kessel E, Pfeiffer RA, Blanke W, Schwarz J. 1978. Terminal to the 5 Mb interval delimited by the two closest deletion of the long arm of chromosome 1 in a malformed BACs flanking the deletion (RP11-529d17 and RP11- newborn. Hum Genet 42:333–337. 100e13): DUSP10 (dual specificity protein phospha- Lesnik Oberstein SA, Kriek M, White SJ, Kalf ME, Szuhai K, den Dunnen JT, Breuning MH, Hennekam RC. 2006. Peters Plus tase 10), TAF1a (TBP-associated factor 1a), DISP1 syndrome is caused by mutations in B3GALTL, a putative (dispatched 1), CAPN2 (calpain 2, larger subunit), glycosyltransferase. Am J Hum Genet 79:562–566. Erratum in: TP53BP2 (tumor protein p53 binding protein 2), NVL Am J Hum Genet 79:985. (nuclear valosin-containing protein-like), WDR26 Mazzeu JF, Pardono E, Ricchieri-Costa A, Kim CA, Brunoni D, (WD repeat domain 26), and DEGS1 (degenerative Martelli L, Lima CEF, Colin G, Vianna-Morgante AM, Otto PA. 2007. Clinical characterization of autosomal dominant and spermatocyte homolog 1). These genes appear as autosomal recessive Robinow syndrome. Am J Med Genet candidates for our patients’ phenotype and may be Part A 143A:320–325. considered as candidates for DRS. Milot E, Fraser P, Grosveld F. 1996. Position effects and genetic The finding of different chromosome rearrange- disease. Trends Genet 12:123–126. Mircher C, Rethore MO, Lespinasse J, Fert-Ferrer S, Lundsteen C, ments in association with DRS points to genetic Kirchoff M. 2003. Interstitial deletion of the short arm of heterogeneity in the syndrome. chromosome 1: Attempt to establish a clinical phenotype (46,XX,del(1)(p22p32)). Am J Med Genet Part A 118A:176–179. Mohammed FMA, Farag TI, Gunawardana SS, Al-Digashim DD, ACKNOWLEDGMENTS Al-Awardi SA, Al-Ohtman SA, Sundareshan TS. 1989. Direct duplication of chromosome 1, dir dup(1)(p21.2 ! p32) in a We thank Mrs. Dalva Marques and Mrs. Ligia S. bedouin boy with multiple congenital anomalies. Am J Med ´ Genet 32:353–355. Vieira for technical assistance, and Maurıcio S. Molina M, Santolaya JM, Saitua G, Hernandez M. 1978. Long-arm Galizia, MD and Luiz Antonio Nunes de Oliveira, deletion of chromosome 1: 46,XY, del (1) (q42). An Esp MD for radiograph analysis. Pediatr 11:729–732. Neu RL, Avila DA, Reddington JM. 1982. A 1q42 deletion in a vietnamese infant. Ann Ge´ne´t 25:154–155. REFERENCES Paganoni S, Anderson KL, Ferreira A. 2004. Differential sub- cellular localization of Ror receptors in Afzal AR, Rajab A, Fenske C, Oldridge M, Elanko N, Terne-Pereira cultured astrocytes. Glia 46:456–466. E, Tu¨ysu¨z B, Murday VA, Patton MA, Wilkie AOM, Jeffery S. Rosenberg C, Janson M, Nordeskjold M, Borresen AL, Vianna- 2000. Recessive Robinow syndrome, allelic to dominant Morgante AM. 1994. Intragenic reorganization of RB1 in a American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

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complex (4;13) rearrangement demonstrated by FISH. Cyto- kinase causes autosomal recessive Robinow syndrome. Nat genet Cell Genet 65:268–271. Genet 25:423–426. Sivasankaran S, Ho NK, Knight L. 1997. De novo interstitial Verma RS, Conte RA, Sayegh SE, Kanjilal D. 1992. The deletion of chromosome 1p with absent corpus callosum–a interstitial deletion of bands q 33-35of long arm of chromo- case report. Ann Acad Med Singapore 26:507–509. some 7: A review with a new case report. Clin Genet 41:82– Tolkendorf E, Hinkel GK, Gabriel A. 1989. A new case of deletion 86. 1q42 syndrome. Clin Genet 35:289–292. Wang LY, Hsu CH, Shih SL, Lin SP. 1997. Robinow syndrome: Turleau C, de Grouchy J, Frezal J, Richardet JM. 1983. Distal 1q report of one case. Chung Hua Min Kuo Hsiao Erh Ko I Hsueh monosomy. 2 new cases and description of the syndrome. Hui Tsa Chih 38:235–238. Ann Genet 26:161–164. Zhao Y, Meng XM, Wei YJ, Zhao XW, Liu DQ, Cao HQ, Liew CC, Van Bokhoven H, Celli J, Kayserili H, Van Beusekom E, Balci S, Ding JF. 2003. Cloning and characterization of a novel cardiac- Brussel W, Skovby F, Kerr B, Percin EF, Akarsu N, Brunner specific kinase that interacts specifically with cardiac troponin HG. 2000. Mutation of the gene encoding the ROR2 tyrosine I. J. Mol. Genet 81:297–304.