A novel PHOX2A/ARIX mutation in an iranian family with congenital fibrosis of extraocular muscles type 2 (CFEOM2)

A novel PHOX2A/ARIX mutation in an iranian family with congenital fibrosis of extraocular muscles type 2 (CFEOM2)
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  A Novel  PHOX2A/ARIX   Mutation in anIranian Family With Congenital Fibrosis of Extraocular Muscles Type 2 (CFEOM2) AHMAD YAZDANI, MD, DANIEL C. CHUNG, DO,MOHAMMAD R. ABBASZADEGAN, P H D, KHOLOUD AL-KHAYER, MD,WAI-MAN CHAN, MILAD YAZDANI, KAZEM GHODSI, P H D,ELIZABETH C. ENGLE, MD, AND ELIAS I. TRABOULSI, MD ●  PURPOSE:  To describe the clinical features of twoaffected members of an Iranian family with autosomalrecessive congenital fibrosis of the extraocular muscles(CFEOM2) and to report their novel mutation in the PHOX2A/ARIX   gene. ●  DESIGN:  Experimental study. ●  METHODS:  SETTING : Institutional practice.  PATIENTPOPULATION : Six members of an Iranian family withCFEOM underwent complete ocular examinations in-cluding assessment of ocular motility, visual acuity,slit-lamp biomicroscopy, tonometry, and ophthalmos-copy.  EXPERIMENTAL PROCEDURE : Mutation analysis of the PHOX2A  gene was performed using polymerase chainreaction amplification of the coding exons and directsequencing of polymerase chain reaction products.  MAINOUTCOME MEASURE : Presence or absence of mutation in PHOX2A  gene in two siblings with exotropia and reces-sive CFEOM. Exotropia and ptosis were corrected surgi-cally in one of the two siblings. ●  RESULTS:  The two affected siblings had bilateral ptosisand exotropia and severe limitation of all extraocularmovements. One patient underwent strabismus surgeryand ptosis repair.  PHOX2A  mutation analysis revealed anovel nonsense mutation in exon 2 (439C 3  T). Bothparents and the unaffected siblings were heterozygous,and the two affected siblings were homozygous for thismutation. ●  CONCLUSION:  The 439C 3  T mutation in this familychanges a glutamine to a stop codon (Q90X) at thebeginning of the PHOX2A homeodomain region. This isthe fourth CFEOM2 mutation in  PHOX2A  and the firstnonsense mutation to be identified. It confirms  PHOX2A as the autosomal recessive CFEOM2 disease gene andprovides evidence that the phenotypic differences be-tween PHOX2A mutations in man and mouse do notresult from hypomorphic PHOX2A alleles in humans.(Am J Ophthalmol 2003;136:861–865. © 2003 byElsevier Inc. All rights reserved.) T HE CFEOM (CONGENITAL FIBROSIS OF THE EXTRA- ocular muscles) syndromes are characterized by con-genital restrictive ophthalmoplegia affectingmuscles in the oculomotor and trochlear nerve distribu-tion. 1 Significant advances have been made in the delin-eation of the genetic types of CFEOM. 1,2 Three clinicalCFEOM phenotypes, CFEOM1, CFEOM2, and CFEOM3have been identified, and three genetic loci,  FEOM1 (Fibrosis of the extraocular muscles 1),  FEOM2 , and FEOM3  have been mapped by linkage analysis. CFEOM1is autosomal dominant, with evidence of mild phenotypicvariability, and typically maps to the  FEOM1  locus in thecentromeric region of chromosome 12 (12p11.2-q12). 3–6 CFEOM3 is also autosomal dominant with variable expres-sion and incomplete penetrance; it maps to the  FEOM3 locus, a 5.6 cM region on 16q24.2-q24.3. 7 Both CFEOM1and CFEOM3 are proposed to result from aberrant devel-opment of the oculomotor nucleus and nerve. 7,8 CFEOM2is autosomal recessive. Individuals with CFEOM2 are bornwith bilateral ptosis and restrictive ophthalmoplegia, withtheir eyes partially or completely fixed in an exotropicposition. This condition was proposed to result fromaberrant development of both the oculomotor and abdu- Accepted for publication Dec 23, 2002.From the Department of Pediatric Ophthalmology and Strabismus andthe Center for Genetic Eye Diseases, Cole Eye Institute, Cleveland ClinicFoundation, Cleveland, Ohio (A.Y., D.C.C., K.A.-K., M.Y., E.I.T.);Mashad University of Medical Sciences, Mashad, Iran (A.Y., M.R.A.,K.G.); and Genetics and Neurology, Boston Children’s Hospital, Har-vard Medical School, Boston, Massachusetts (W.-M.C., E.C.E.).Supported by Foundation Fighting Blindness, N.Y. (Dr. Traboulsi), andby grant ROI EY12498 (Dr. Engle).Inquiries to Elias I. Traboulsi, MD, Cole Eye Institute, The ClevelandClinic Foundation, i32 9500 Euclid Avenue, Cleveland, OH 44195; fax:(216) 445–2226; e-mail: ©  2003 BY  E LSEVIER  I NC . A LL RIGHTS RESERVED . 0002-9394/03/$30.00  861 doi:10.1016/S0002-9394(03)00891-2  cens nuclei and nerves. 9 The CFEOM2 gene was mappedto the  FEOM2  locus on chromosome 11q13. 9 Traboulsiand coworkers 10 presented evidence of genetic heteroge-neity in CFEOM2, with possible allelism to CFEOM1 insome families. Nakano and colleagues 11 identified muta-tions in the homeobox transcription factor  PHOX2A  (alsoknown as  ARIX ) in one Turkish and three Saudi Arabianfamilies whose CFEOM2 phenotype mapped to the FEOM2  locus. Two of the mutations identified by Nakanoand coworkers 11 are predicted to disrupt splicing: a G 3  Amutation at position  1 of the GT splice donor sequenceof exon 1 and a G 3  A mutation at position –1 of the AGsplice acceptor site of exon 3; a third mutation, a C 3  Tmissense mutation at nucleotide 386 in exon 1 results in anarginine to valine substitution at codon 72, altering theconserved brachyury-like domain.In mice and zebrafish the homeodomain transcriptionfactor  phox2a  is essential to the development of cranialnerve nuclei III and IV, the locus coeruleus, and selectiveparasympathetic and placode-derived cranial sensory gan-glia; mice lacking the  phox2a  homeodomain die atbirth. 12–15 The CFEOM2 phenotype in man appears to belimited to the ocular findings described above. Nakano andcoworkers 11 proposed that the phenotypic differences be-tween man and mouse with  PHOX2A /  phox2a  mutationscould result either from functional differences between thehighly related human and mouse genes or because thethree CFEOM2 mutations they identified represent hypo-morphic alleles of   PHOX2A , resulting in the rescue of PHOX2A function in adrenergic and cranial sensory, butnot motor, neurons.We report a novel nonsense mutation in the PHOX2Agene in an Iranian family with CFEOM2 that supports thefirst of these two hypotheses. METHODS SIX MEMBERS OF A CONSANGUINEOUS FAMILY WITH CFEOM2, including one affected son and one affecteddaughter, underwent detailed ocular examinations. Hori-zontal muscle surgery and ptosis repair were performed onthe affected daughter. Informed consent was obtained andblood samples were collected from all family members.Lymphocytic DNA was extracted according to standardprocedures.Mutation analysis of the  PHOX2A  gene was performedon all DNA samples using primers previously published. 11 For standard polymerase chain reaction (PCR) amplifica-tion, we used Master Amp PCR Optimization Kit (Epicen-ter Technologies, Madison, Wisconsin, USA). PCR 1xPreMix for 10   l reaction contained 1.5 mM MgCl 2  and6x Master Amp Enhancer for exon 1a, 2.5 mM MgCl 2  and6x Master Amp Enhancer for exon 3b, 3.5 mM MgCl 2  and6x Master Amp Enhancer for exon 1b and 3a, 1.5 mMMgCl 2  and 8x Master Amp Enhancer for exon 2, 0.5 mMof each primer, 50 ng of genomic DNA and 0.5 unit of QIAGEN (QUIAGEN Inc., Valencia, California, USA)Taq DNA polymerase. We carried out PCR amplificationwith initial denaturation at 95 C for 10 minutes, followedby 35 cycles at 95 C for 30 seconds, at 50 C to 65 C for 30seconds, and at 72 C for 40 seconds, and final extension at72 C for 10 minutes. For each GC-rich region (exon 1aand 3b), we carried out PCR amplification with an initialdenaturation at 95 C for 15 minutes, followed by 35 cyclesat 94 C for 40 seconds, at 62 C to 68 C for 1 minute andat 72 C for 1 minute, and final extension at 72 C for 10minutes. Polymerase chain reaction products were run onagarose gel, cut out, and purified on amicon purificationcolumns and used as templates for direct sequencing. 11 RESULTS THE AFFECTED SON (FIGURE 1, TOP) AND DAUGHTER IN this family had classical clinical findings of CFEOM2. Theeyes were fixed in an exotropic position with bilateralsevere ptosis. The angle of the exotropia was larger than  FIGURE 1. (Top) Brother with bilateral ptosis and exotropia.The patient is using his forehead to elevate upper lids. (Bottom)Affected sister after bilateral lateral rectus recession and browsuspension. There is marked improvement in ptosis but signif-icant residual exotropia. A MERICAN  J OURNAL OF  O PHTHALMOLOGY 862  N OVEMBER 2003  80  in the girl and 70  in the boy. There were minimalabduction movements. The pupils were miotic with noreaction to light bilaterally in both cases. The girl wasmyopic with a refractive error of –3.00  0.50  90 in theright eye, and –2.50  0.50  100 in the left eye. The boywas hypermetropic with a refractive error of    5.00  1.00  30 in the right eye, and  4.00  1.50  120 in theleft eye. The girl had bilateral amblyopia presumed to befrom a combination of ptosis and strabismus; her visualacuity was 20/60 in the right eye and counting fingers at 2meters in the left eye. The Bell phenomenon was absent,and there was mild bilateral exposure keratopathy in bothpatients. Recession of both lateral rectus muscles andresection of both medial rectus muscles were performed onthe girl with only a modest change in the position of theeyes and residual exotropia (Figure 1, bottom). Forcedduction testing at the time of surgery revealed significantlimitation to movement of both eyes in all directions.Bilateral brow suspension was performed for ptosis repair,and to prevent exposure keratopathy, the upper lids werebrought only to a level 1 mm above the pupil in primaryposition. The boy did not undergo any surgical procedures.Both parents and the other two sons had no abnormalocular findings. The affected children had no evidentdisorder of sleep or mood and denied dryness of theirmouth and nasal passages. They had normal taste. PHOX2A  gene analysis in this family showed homozy-gosity for a 439C 3  T (CAG 3  TAG) mutation in PHOX2A  exon 2 in both affected siblings (Figure 2). Theparents and two unaffected brothers were heterozygous forthe mutation. This mutation results in a change from aglutamine to a stop codon (Q90X). DISCUSSION CONGENITAL FIBROSIS OF EXTRAOCULAR MUSCLES TYPE 2 is an autosomal recessive disease characterized by exotropicophthalmoplegia and bilateral blepharoptosis. The ocularmotor defect is congenital and nonprogressive. The phe-notype we report in this Iranian CFEOM2 pedigree is thesame as that in the reported Turkish and Saudi CFEOM2families. 9 In addition to the presumed maldevelopment of the oculomotor and trochlear nuclei, we believe that thesmall pupils in our two patients and in some of the patientsin the Saudi Arabian families may result from abnormali-ties in the noradrenergic autonomic nervous system thatinnervates the dilator muscles of the iris. Extensive search  FIGURE 2. Mutation analysis of  ARIX   by direct automated sequencing in a control unaffected individual (top), a heterozygouscarrier parent (middle) and a homozygous affected child (bottom) from the pedigree. The mutated 439C 3  T nucleotide is indicatedby the black arrow and the complete nucleotide and corresponding amino-acid sequences are shown below the affected sequence,with the mutated base and stop codon indicated in red. ARIX   M UTATION IN CFEOM V OL .  136 , N O .  5  863  for additional clinical or subclinical abnormalities resultingfrom locus coeruleus or other choroidal neovascularizationor peripheral autonomic nervous system structures thatmay depend on PHOX2A for normal development has notbeen undertaken in this family. However, they did nothave any evident disorder of sleep or mood and they didnot have abnormalities of their lacrimal, nasal or salivarysecretions. We followed previous recommendations of clinical management of patients with CFEOM in one of our patients and performed large recessions of tight musclesand correction of ptosis using brow suspension. 16 Correc-tion of refractive errors and aggressive amblyopia manage-ment should be initiated early in life optimize vision andprevent amblyopia. 16 The exotropia in one of our twopatients did not respond well to the recommended eyemuscle surgical procedures. The ptosis was corrected withbilateral brow suspension.The mutation that we believe is causing the disease inthis consanguineous pedigree is a  PHOX2A  439C 3  Tchange that results in a stop (Q90X) mutation (Figure 3).This is the fourth CFEOM2 mutation in  PHOX2A  and thefirst nonsense mutation to be identified. This nonsensemutation will truncate the  PHOX2A  message beyond thethird amino acid of the homeodomain and is remarkablysimilar to the homozygous mutation generated in  phox2a   /   mice, which deletes the entire mouse homeodo-main and all downstream coding sequences (Figure 3). 12 These two mutations could result in unstable mRNAs or intruncated NH2-terminal PHOX2A/phox2a peptides. Bothmutations are predicted to act as null mutations, becausethe homeodomain is deleted and the homozygous  phox2a mouse mutants do not have detectable message whenprobed with 5’  phox2a  cRNA. 12 The human CFEOM2 phenotype appears to be limitedto the ocular motility and pupillary abnormalities found inthis and the previously reported families. In contrast,mouse  phox2a  is not only essential to oculomotor andtrochlear nuclear development, but also to the normaldevelopment of parasympathetic and sensory ganglia of thehead and for survival beyond birth. The cause of thesephenotypic differences between human  PHOX2A  andmouse/zebrafish  Phox2a  mutations are not known. Thefinding that the affected members of this CFEOM2 familyharbor a  PHOX2A  nonsense mutation virtually indistin-guishable from the  phox2a    /   null mouse mutation,however, suggests that the phenotypic differences areunlikely to result from the CFEOM2 mutations function-ing as hypomorphic alleles of   PHOX2A . Instead, the pheno-typic differences are more likely to result from unidentifiedfunctional differences between the highly related human andmouse genes. Learning more about these functional differ-ences should provide insight into the role that PHOX2Aplays in oculomotor and trochlear development and themolecular basis of complex strabismus syndromes.  FIGURE 3. Schematic representation of  ARIX   genomic organization with the localization of the mutations identified to date. Thehorizontal line denotes the intronic regions. The boxes indicate exons 1–3 with coding regions indicated by filled boxes and 5’ and3’ untranslated regions indicated by empty boxes. The gray texture-filled boxes below exons 2 and 3 indicate the sequence thatencodes the ARIX homeodomain. Note that the locations of the start of the  arix   /   mouse deletion and the human nonsensemutation are adjacent at the 5’ end of the homeodomain. A MERICAN  J OURNAL OF  O PHTHALMOLOGY 864  N OVEMBER 2003  REFERENCES 1. Engle EC. The genetics of strabismus: Duane, Mobius, andfibrosis syndromes: In: Traboulsi EI, editor: Genetic diseasesof the eye. New York: Oxford University Press, 1999:477–512.2. Engle EC. Applications of molecular genetics to the under-standing of congenital ocular motility disorders. Ann N YAcad Sci 2002;956:55–63.3. Engle EC, Kunkel LM, Specht LA, Beggs AH. Mapping agene for congenital fibrosis of the extraocular muscles to thecentromeric region of chromosome 12. Nat Genet 1994;7:69–73.4. Engle EC, Marondel I, Houtman WA, et al. Congenitalfibrosis of the extraocular muscles (autosomal dominantcongenital external ophthalmoplegia): genetic homogeneity,linkage refinement, and physical mapping on chromosome12. Am J Hum Genet 1995;57:1086–1094.5. Sener EC, Lee BA, Turgut B, Akarsu AN, Engle EC. Aclinically variant fibrosis syndrome in a Turkish family mapsto the CFEOM 1  locus on chromosome 12. Arch Ophthalmol2000;118:1090–1097.6. Engle EC, McIntosh N, Yamada K, et al. CFEOM1, theclassic familial form of congenital fibrosis of the extraocularmuscles, is generally heterogeneous but does not result frommutations in ARIX. BMC Genet 2002;3:3.7. Doherty E, Macy M, Wang S, et al. CFEOM3: a newextraocular congenital fibrosis syndrome that maps to16q24.2-q24.3. Inv Ophthalmol Vis Sci 1999;40:1687–1694.8. Engle EC, Goumernov B, McKeown CA, et al. Oculomotornerve and muscle abnormalities in congenital fibrosis of theextraocular muscles. Ann Neurol 1997;41:314–325.9. Wang S, Zwaan J, Mullaney P, et al. Congenital fibrosis of the extraocular muscles type 2 (CFEOM2), an inheritedexotropic strabismus fixus, maps to distal 11q13. Am J HumGenet 1998;63:517–525.10. Traboulsi EI, Lee BA, Mousawi A, Khamis AR, Engle EC.Evidence of genetic heterogeneity in autosomal recessivecongenital fibrosis of the extraocular muscles. Am J Oph-thalmol 2000;129:658–662.11. Nakano M, Yamada K, Fain J, et al. Homozygous mutationsin ARIX (PHOX2A) result in congenital fibrosis of theextraocular muscles type 2. Nat Genet 2001;29:315–320.12. Morin X, Cremer H, Hirsch MR, Kapur RP, Goridis C,Brunet JF. Defects in sensory and autonomic ganglia andabsence of locus coeruleus in mice deficient for the ho-meobox gene Phox2a. Neuron 1997;18:411–423.13. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF.Expression and interactions of the two closely related ho-meobox genes Phox2a and Phox2b during neurogenesis.Development 1997;124:4065–4075.14. Guo S, Brush J, Teraoka H, et al. Development of noradren-ergic neurons in the zebrafish hindbrain requires BMP, FGF8,and the homeodomain protein soulless/Phox2a. Neuron1999;24:555–566.15. Adachi M, Lewis EJ. The paired-like homeodomain protein,Arix, mediates protein kinase A-stimulated dopamine beta-hydroxylase gene transcription through its phosphorylationstatus. J Biol Chem 2002;277:22915–22924.16. Traboulsi EI, Jaafar M, Kattan H, Parks MM. Congenitalfibrosis of the extraocular muscles: report of 24 cases illus-trating the clinical spectrum and surgical management. AmOrthopt J 1993;43:45–53. ARIX   M UTATION IN CFEOM V OL .  136 , N O .  5  865
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