Horizontal gaze palsy and progressive scoliosis due ...

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... of Florida, Jacksonville, Florida, USA,. 3Division of Genetics, Maulana Azad Medical College, New Delhi, India, 4Saad Specialist Hospital, Al-Khobar, Saudi.

Ophthalmic Genetics, 32(4), 231–236, 2011 Copyright © 2011 Informa Healthcare USA, Inc. ISSN: 1381-6810 print/ 1744-5094 online DOI: 10.3109/13816810.2011.580445

RESEARCH REPORT

Horizontal gaze palsy and progressive scoliosis due to a deleterious mutation in ROBO3 Khaled K. Abu-Amero1,2, Seema Kapoor3, Ali Hellani4, Sumit Monga5, and Thomas M. Bosley1,6 Ophthalmic Genetics Laboratory, Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia, 2Department of Ophthalmology, College of Medicine, University of Florida, Jacksonville, Florida, USA, 3 Division of Genetics, Maulana Azad Medical College, New Delhi, India, 4Saad Specialist Hospital, Al-Khobar, Saudi Arabia, 5Pediatric Ophthalmology Service, Shroff Eye Centre, New Delhi, India, and 6Division of Neurology, Cooper University Hospital, Camden, NJ, USA

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ABSTRACT Purpose: To describe a family with horizontal gaze palsy and progressive scoliosis with a deleterious mutation in the ROBO3 gene. Methods: All family members had full ophthalmologic, neurologic, and orthopedic examinations and complete sequencing of the ROBO3 gene. Results: Four affected members had complete loss of horizontal gaze with progressive scoliosis that varied between family members. ROBO3 sequencing revealed a novel 15 base deletion (c.2_16 delTGCTGCGCTACCTGC) in exon 1 that segregated in homozygous form with the phenotype and probably alters the shape and ionic charge of the extracellular immunoglobulin motif 1. This mutation was not detected in 100 control chromosomes. Conclusions: The novel ROBO3 mutation in this family may be among the most deleterious yet reported. Family members in general were severely affected, but comparison of this family to other families with ROBO3 mutations did not yield a definitive phenotype-genotype correlation. Keywords:  horizontal gaze palsy and progressive scoliosis, HGPPS, ROBO3, scoliosis, brainstem development

INTRODUCTION

the majority of which were inherited in homozygous status in consanguineous families.6,7 We describe an Afghani family examined in New Delhi, India, with the ocular motility and spinal abnormalities of HGPPS in four out of five children caused by a particularly deleterious mutation in ROBO3.

Horizontal gaze palsy with progressive scoliosis (HGPPS; OMIM 607313) is a rare, autosomal recessive disorder1 characterized by the congenital absence or severe restriction of horizontal gaze,2 progressive scoliosis beginning in early childhood currently treatable only by spine surgery,3 and failure of corticospinal and somatosensory neuronal tracts to decussate in the medulla.4 HGPPS is caused by mutations of the ROBO3 gene, which encodes a protein that shares homology with the roundabout family of transmembrane receptors important in axon guidance and neuronal migration, including decussation of developing nerve fiber tracts in the brainstem.5 To date, there are 24 mutations reported in individuals with HGPPS,

MATERIALS AND METHODS Clinical Examinations This consanguineous Afghani family sought evaluation because of defective eye movements and scoliosis in four out of five children. The four affected children were males aged 14 (II-1), 12 (II-2), 11 (II-3),

Received 14 January 2011; revised 31 March 2011; accepted 05 April 2011 Correspondence: Khaled K. Abu-Amero, PhD, Ophthalmic Genetics Laboratory, Department of Ophthalmology, College of Medicine, King Saud University, PO Box 245, Riyadh 11411, Saudi Arabia. Email: [email protected]; Tel: +96614786100, Fax: +96614775742.

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232    K. K. Abu-Amero et al. and 8 years (II-4) at the time of complete ophthalmologic, neurologic, and orthopedic examinations (see pedigree Figure 1). All children had spine x-rays, and brain MRI was performed on Patient II-3. The unaffected parents (I-1 and I-2) and one unaffected female child (II-5) were also examined.

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Genetic analysis After the father signed informed consent, genomic DNA was extracted from peripheral blood of all family members using the PURGENE® kit from Gentra Systems (Minneapolis, MN, USA), quantified spectrophotometrically, and stored at −20°C in aliquots until required. All 28 coding exons and exon-intron boundaries of the ROBO3 gene were amplified as previously described.6 Each successfully amplified fragment was directly sequenced with the same primers used for PCR amplification.6 Sequencing was carried out with the reverse primers used for amplification together with the BigDye® terminator v3.1 kit (Applied Biosystems). Samples were run on the ABI 3130xl genetics analyzer (Applied Biosystems) according to the manufacturer protocol. The sequence obtained was compared to ROBO3, the Homo sapiens roundabout axon guidance receptor homolog 3 (Drosophila) mRNA (GenBank NM_022370).

Prediction of the effect of the mutation on the protein structure The wildtype and mutated ROBO3 proteins were modeled using Lasergene Protean software (DNASTAR, Inc., Wisconsin, USA), which analyzes protein structure using a diverse selection of factors, including secondary structure according to the formula of Parry,8 hydropathy according to the method of Kyte-Doolittle,9 antigenicity according to the method of Rothbard and Taylor,10,11 amphilicity according to the method of Eisenberg,12 and flexibility of the protein according to the KarplusSchultz method.13

RESULTS Clinical observations Antenatal and birth history were unremarkable for the four affected children, but motor milestones were slightly delayed. In each patient, ocular motility restriction was noted shortly after birth. Progressive scoliosis was diagnosed in early childhood in the two most affected children (II-3 and II-4) but was only diagnosed on orthopedic examination at ages 14 years (II-1) and 12 years (II-2) in the two less affected children.

Ophthalmologic examination of anterior segment, ocular fundus, pupils, and intraocular pressure were normal in all four affected boys. All were hyperopic with corrected visual acuity varying between 6/6 OU and 6/18 OU (in II-4, the youngest). Horizontal ductions and versions were completely absent in all affected children (Figure 2A, 2B, and 2C) with full vertical eye movements. All patients had a mild esotropia requiring a slight head turn in order to fixate with either eye. Patients II-1, II-2, and II-4 had small amplitude horizontal pendular nystagmus in both eyes that was unchanged by vertical gaze or attempted horizontal gaze. Convergence was absent in Patient II-3, but Patients II-1 and II-2 made convergence movements on attempted lateral gaze to either side (Figure 2D and 2E) compatible with synergistic convergence.14 Each individual turned his head to either side in order to refixate horizontally. Patient II-3 had marked kyphoscoliosis (Figure 2F) with wedging and rotation of the vertebrae on x-ray, while Patients II-1 and II-2 had moderate scoliosis, and II-4 (the youngest) had mild scoliosis confirmed by spine x-rays. Brain MRI in Patient II-3 revealed pontine hypoplasia with an anterior midline cleft in the medulla and pons.1,15 CSF spaces were prominent anterior to the frontal lobes with widened Sylvian fissures suggesting benign external hydrocephalus. All affected children were cognitively normal with no other focal neurologic deficits. Father (I-1), mother (I-2), and the unaffected sister (II-5) had normal ocular motility without scoliosis.

Genetic results Sequencing the full ROBO3 gene revealed the presence of a 15 base deletion in exon 1 of the ROBO3 gene (c.2_16 delTGCTGCGCTACCTGC) that was inherited in homozygous status in all affected individuals (II-1, II-2, II-3 and II-4) and in heterozygous status in both parents (I-1 and I-2) and their unaffected daughter (II-5). It was not detected in 50 unrelated, healthy individuals (100 chromosomes) of matching ethnicity. This deletion mutation removes five amino acids from the immunoglobulin like motif 1 (Ig1), shortening the first alpha region, shifting the positions of subsequent alpha and beta regions and protein turns in Ig1, and changing the hydrophobicity of this extracellular segment of the protein 9 (Figure 3A, 3B, and 3C). Additionally, a flexible region was lost in the mutated ROBO3 protein, changes were observed in the alpha and beta amphipathic regions and surface probability plot was altered in the mutated protein.

DISCUSSION Four of five children of this consanguineous family had the typical features of HGPPS,1 including complete Ophthalmic Genetics

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Deleterious ROBO3 mutation    233

FIGURE 1.  Pedigree and ROBO3 deletion. Pedigree of the consanguineous Afghani family. Chromatogram of sequencing results for exon 1 of the ROBO3 gene. Chromatogram (A) shows the reverse sequence detected in normal control. Chromatogram (B) shows sequences from individuals II-1, II-2, II-3 and II-4 who were homozygous for the deletion. The arrow indicates the deletion position. Chromatogram (C) shows the reverse sequence detected in carrier individuals (I-1, I-2 and II-5) who were heterozygous for the deletion. The sequence after the deletion shows double peaks as it is reading the normal and the deleted allele at the same time. For clarity, the reverse sequence is shown here and the deleted sequence in the forward position is 5′− TGC TGC GCT ACC TGC -3′ which is the reverse complement of the sequence shown.

horizontal gaze restriction and progressive scoliosis beginning in early childhood that was particularly severe in Patient II-3 (Figure 2F). Three had congenital nystagmus,1 and two had synergistic convergence, an unusual ocular motility pattern reported in HGPPS in which adduction is substituted for abduction on © 2011 Informa Healthcare USA, Inc.

attempted lateral gaze to either side.14 Brain MRI on Patient II-3 revealed brainstem hypoplasia of the lower brainstem typical of HGPPS.1,15 This family had a 15 base deletion in exon 1 of the ROBO3 gene that segregated appropriately with the disease phenotype because all affected individuals

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234    K. K. Abu-Amero et al.

FIGURE 2.  Clinical features. (A–C) Horizontal ocular motility of Patient II-3 illustrating primary gaze (B), right gaze (A), and left gaze (C); (D, E) Horizontal ocular motility of Patient II-2 in primary gaze (D) and right gaze (E) showing synergistic convergence on attempted lateral gaze; and (F) severe scoliosis of Patient II-3.

FIGURE 3.  ROBO3 protein. Schematic presentation of the structure of the (A) wildtype and (B) mutant ROBO3 protein created by the Lasergene Protean software (see Methods). The changes in the alpha and beta regions are boxed. The red arrow points to the loss of the flexible region and the dark green arrow indicates the changes in the surface probability plot. (C) The hydrophobicity index by the method of Kyte and Doolittle, where a score of 4.6 is the most hydrophobic and a score of −4.6 is the most hydrophilic. The top line presents the wildtype ROBO3 protein with amino acids and their corresponding hydrophobicity index (numbers in parentheses represent the location of the amino acid, i.e. position 1 means AA 1 on the codon). The bottom line represents the corresponding amino acids in the mutated ROBO3 protein together with their hydrophobicity index.

were homozygous, while the parents and the one unaffected child were heterozygous. The mutation is located in the Ig1 domain and results in the deletion of five amino acids and a substantial abnormality in the conformation and ionic charge of this extracellular component of the resultant protein. In comparison

to the other 24 ROBO3 mutations reported thus far (Figure  4), this 15 base deletion in exon 1 is likely among the most deleterious because it involves a big deletion. Therefore, this mutation may offer an opportunity to investigate again whether HGPPS phenotypic expression may correlate with the expected Ophthalmic Genetics

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Deleterious ROBO3 mutation    235

FIGURE 4.  Physical map of ROBO3 domains with location of reported mutations. The 24 reported mutations in the ROBO3 are indicated on a physical map of the ROBO3 domains. Missense mutations are listed above the diagram whereas nonsense and frameshift mutations are listed below.

deleterious effect of this ROBO3 mutation on the protein function. In general, phenotype-genotype correlations have not been obvious in HGPPS, possibly because of intrafamilial variability of the cardinal features and because estimating the underlying severity of both components of such a correlation is difficult.1,5,6 For example, a genotypic assessment of ROBO3 mutations presents challenges because relatively little is known about the function of various ROBO3 domains or actions of alternative splice forms of ROBO3 in the human brainstem.5,16 Ocular motility assessment is dependent at least in part on patient effort, and the variability of horizontal gaze restriction in HGPPS (ranging only from almost complete to complete) is limited. All four affected individuals in the current family had complete restriction of horizontal gaze, two with an esotropia and three with horizontal pendular nystagmus. Comparing ocular motility in this family to available clinical data regarding genotyped individuals reported previously from Saudi Arabia1,6,14 revealed that the frequency of complete horizontal gaze restriction, esotropia, and nystagmus were not significantly different from this family, but the variability within both groups was small. Scoliosis has a greater range of variability than ocular motility in HGPPS and offers a somewhat better opportunity to quantify one clinical aspect of the syndrome. However, scoliosis is well documented to change over time.1,6 Many patients with HGPPS have severe scoliosis,5 including Patient II-3 in the current family (Figure 2F). However, the other three affected members (II-1, II-2, and II-4) had less spinal curvature, highlighting intra-familial variability rather than severity of scoliosis in this family. One other reported family17 (also examined by TMB), had minimal scoliosis with complete horizontal gaze palsy in two affected brothers more than 20 years old who were never treated. This family had a missense mutation © 2011 Informa Healthcare USA, Inc.

in exon 2 affecting Ig1-2, a considerably less profound mutation than in the current family. However, two other reported families had missense mutations in exon 2 associated with substantially worse scoliosis. It is possible that these mutations in exon 2 have differential effects on protein folding or trafficking despite being close to each other. Nevertheless, a more likely interpretation at the moment is that scoliosis in HGPPS patients is variable according to genetic, epigenetic, and/or environmental factors that are at least in part independent of the specific mutation in ROBO3. Currently, therefore, a phenotype-genotype correlation in HGPPS is not apparent, but may be subject to our limited ability to assess each patient with this disorder both genetically and clinically. Most reported ROBO3 mutations are scattered throughout the extracellular portion of the gene, with the only recognized intracellular mutation thus far being an insG (c.3325 + 1G) mutation in exon 23.5 Without additional knowledge of ROBO3 function in humans16 it is not yet clear whether these mutations alter ligand recognition, protein folding, or targeting, and whether resultant changes in protein function might have a differential effect on developing nerve fiber tract decussation and/or on clinical expression.

ACKNOWLEDGMENTS Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

REFERENCES 1.

Bosley TM, Salih MA, Jen JC, et  al. Neurologic features of horizontal gaze palsy and progressive scoliosis with mutations in ROBO3. Neurology 2005;64(7):1196–203.

236    K. K. Abu-Amero et al.

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2.

Sharpe JA, Silversides JL, Blair RD. Familial paralysis of horizontal gaze associated with pendular nystagmus, progressive scoliosis, and facial contraction with myokymia. Neurology 1975;25(11):1035–1040. 3. Dretakis EK. Congenital horizontal gaze palsy and kyphoscoliosis. J Med Genet 1980;17(4):324. 4. MacDonald DB, Streletz LJ, Al-Zayed Z, et al . Intraoperative neurophysiologic discovery of uncrossed sensory and motor pathways in a patient with horizontal gaze palsy and scoliosis. Clin Neurophysiol 2004;115(3):576–582. 5. Jen JC, Chan WM, Bosley TM, et al . Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis. Science 2004;304(5676):1509–1513. 6. Abu-Amero KK, al Dhalaan H, al Zayed Z, et al. Five new consanguineous families with horizontal gaze palsy and progressive scoliosis and novel ROBO3 mutations. J Neurol Sci 2009;276(1–2):22–26. 7. Amoiridis G, Tzagournissakis M, Christodoulou P, et  al . Patients with horizontal gaze palsy and progressive scoliosis due to ROBO3 E319K mutation have both uncrossed and crossed central nervous system pathways and perform normally on neuropsychological testing. J Neurol Neurosurg Psychiatry 2006;77(9):1047–1053. 8. Parry DA. Coiled-coils in alpha-helix-containing proteins: analysis of the residue types within the heptad repeat and the use of these data in the prediction of coiled-coils in other proteins. Biosci Rep 1982;2(12):1017–1024.

9. 10. 11. 12. 13.

14. 15. 16. 17.

Kyte J, Doolittle RF. A simple method for ­displaying the hydropathic character of a protein. J Mol Biol 1982;157(1):105–132. Magnan CN, Zeller M, Kayala MA, et al. High-throughput prediction of protein antigenicity using protein microarray data. Bioinformatics; 26(23):2936–2943. Rothbard JB, Lechler RI, Howland K, et al . Structural model of HLA-DR1 restricted T cell antigen recognition. Cell 1988;52(4):515–523. Eisenberg D, Wilcox W, McLachlan AD. Hydrophobicity and amphiphilicity in protein structure. J Cell Biochem 1986;31(1):11–17. Zoete V, Michielin O, Karplus M. Relation between sequence and structure of HIV-1 protease inhibitor complexes: a model system for the analysis of protein flexibility. J Mol Biol 2002;315(1):21–52. Khan AO, Oystreck DT, Al-Tassan N, et al . Bilateral synergistic convergence associated with homozygous ROB03 mutation (p.Pro771Leu). Ophthalmology 2008;115(12):2262–2265. Pieh C, Lengyel D, Neff A, et al . Brainstem hypoplasia in familial horizontal gaze palsy and scoliosis. Neurology 2002;59(3):462–463. Evans TA, Bashaw GJ. Functional diversity of Robo receptor immunoglobulin domains promotes distinct axon guidance decisions. Curr Biol 2010;20(6):567–572. Amouri R, Nehdi H, Bouhlal Y, et al . Allelic ROBO3 heterogeneity in Tunisian patients with horizontal gaze palsy with progressive scoliosis. J Mol Neurosci 2009;39(3):337–341.

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