A Novel Mutation in the GJA1 Gene in a Family with Oculodentodigital Dysplasia

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: JANEY L. WIGGS, MD, PhD A Novel Mutation in the GJA1 Gene in a Family With Oculodentodigital Dysplasia

José P. C. Vasconcellos, MD, PhD; Mônica B. Melo, PhD; Rui B. Schimiti, MD, PhD; Norisvaldo C. Bressanim, MD; Fernando F. Costa, MD, PhD; Vital P. Costa, MD, PhD

Objectives: To describe a Brazilian family with oculo- closure glaucoma, and 1 had open-angle glaucoma. A new dentodigital dysplasia (ODDD) and to screen for muta- mutation in the GJA1 gene was identified, consisting of tions in the gap junction protein alpha 1 (GJA1) gene in a change from proline to histidine at codon 59. This mu- this family. tation segregated through members with the ODDD phenotype. Analysis of the control group by means of re- Methods: Twelve members of a 3-generation family with striction fragment length polymorphism (MvaI enzyme) ODDD underwent screening for mutations of the GJA1 gene did not disclose this mutation. and a comprehensive ophthalmic examination. We defined ODDD on the basis of clinical characteristics described in Conclusion: Our results demonstrate a new mutation this syndrome (microdontia, caries, enamel hypoplasia, thin (P59H) in the GJ1A gene, identified in a family with nose,andsyndactyly)andeyeabnormalitiessuchasmicroph- ODDD syndrome. thalmos, iris atrophy, and glaucoma. Direct sequencing of the GJA1 gene was performed using DNA collected from Clinical Relevance: The presence of different forms peripheral blood. A control group of 60 healthy individu- of glaucoma in families with ODDD may indicate a new als underwent evaluation by means of enzyme digestion. mutation in the GJA1 gene. Results: Among the 8 members of this family who were characterized as having ODDD, 2 showed chronic angle- Arch Ophthalmol. 2005;123:1422-1426

CULODENTODIGITAL DYS- fourth and fifth fingers and/or the third or plasia (ODDD) is a syn- fourth toes, camptodactyly of the fifth fin- drome characterized by ger, midphalangeal hypoplasia or aplasia of malformations that in- 1 or more digits or toes, and other abnor- volve the face, eyes, teeth, malities such as a mandible with a wide al- andO bones and by neurological alterations.1 veolar ridge and broad tubular bones.13 Neu- Although sporadic cases are reported in the rological deficits have been reported in a few literature (most of them involving elderly studies, including spastic paraparesis, qua- parents), ODDD is inherited in an autoso- driparesis, gait disturbance, neurogenic mal dominant pattern with high penetrance bladder disturbances, ataxia, hearing loss, andvariableexpression.2 Thisconditionwas dysarthria, and seizures.4 Magnetic reso- first described in 1920 by Lohmann3 in 2 nance imaging shows diffuse abnormali- Author Affiliations: patients with bilateral camptodactyly of the ties of the subcortical cerebral white mat- Departments of Ophthalmology fifth finger and microphthalmos.4 In 1957, ter that have been suggested as the cause of (Drs Vasconcellos, Schimiti, and 5 4,14 V. P.Costa) and Clinical Meyer-Schwickerath et al introduced the neurological manifestations. Other char- Medicine–Hemocentro term ODDD to describe 2 patients with mal- acteristics that may be found in ODDD are (Dr F.F.Costa), State University formations in the eyes, including bilateral depressed nasal bridge, thin nose with hy- of Campinas, Campinas, Brazil; microphthalmoswithglaucomaandanoma- poplastic alae nasi, small anteverted nares, Department of Physiological lies of the iris, nose, teeth, and bones, and cleft palate.13 Sciences, Santa Casa de São whereas in 1963, Gorlin et al6 subsequently The ocular manifestations of ODDD Paulo, São Paulo, Brazil defined it as a syndrome. Since then, sev- may involvetheentireocularglobe(microph- (Dr Melo); Cascavel University eral authors reported this condition in more thalmos), associated with anterior segment Hospital, UNIOESTE (State than 240 individuals.4,7-13 dysgenesis.7,8 Otherwise,theaxiallengthmea- University of the West of Paraná), Cascavel, Brazil The teeth anomalies observed in ODDD surementsmaybenormalandtheabnormali- (Dr Bressanim); and include microdontia, caries, enamel hypo- ties restricted to the anterior segment (iris Department of Ophthalmology, plasia, and partial anodontia. The skeletal atrophy, remnants of the pupillary mem- University of São Paulo, São malformations occur mainly in the extremi- brane, iridoschisis, and cataract).9 Posteri- Paulo, Brazil (Dr V. P.Costa). ties and consist of syndactyly involving the or segment abnormalities include remnants

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 of the hyaloid artery and an increased number of retinal 9 vessels at the optic disc. Other less frequent ocular I Individuals Without ODDD features of ODDD are nystagmus, palpebral fissure hy- 12 8 Individuals poplasia, epicanthal folds, and convergent strabismus. With ODDD Glaucoma is one of the causes of visual loss in ODDD. Open-angle and angle-closure glaucomas have been reported in eyes with ODDD, as well as goniodysgenesis.8-13 II 14 12 3 456 In 1997, Gladwin et al evaluated ODDD in 6 fami- 44/+ 42/+ 37/+ lies (autosomal dominant pattern) using linkage analysis. The authors identified the locus associated with ODDD III 1 2 3 4 5 at chromosome 6q22-q24 in a 28-centimorgan (cM) re- 22/− 16/− 19/− 16/− 8/− gion, delimited by markers D6S474 proximally and D6S292 distally. Boyadjiev et al2 performed 2-point link- Figure 1. Pedigree of a family with oculodentodigital dsyplasia (ODDD) age analysis in 7 families with ODDD and significantly syndrome harboring the proline→histidine mutation at codon 59. The age at reduced the size of the critical region (11 cM in male and last examination is shown by each symbol. Individual I:1 died with a history 15 of glaucoma and blindness. Circle indicates female family member; square, 20 cM in female subjects). Subsequently, Paznekas et al ϩ identified structural changes in the gene responsible for male family member; −, absence of glaucoma; and , presence of glaucoma. synthesis of connexin 43 (Cx43), the gap junction protein alpha 1 (GJA1) gene, in all 17 families with ODDD autosomal dominant inheritance pattern (Figure 1). The who underwent screening for mutations in this gene. affected individuals had the classical characteristics de- The goals of this study were to describe a Brazilian fam- scribed in ODDD, including microdontia, caries, enamel ily with ODDD and to screen for mutations in the GJA1 hypoplasia, syndactyly involving the second and third toes, gene in this family. and mild clinodactyly (Figure 2). These individuals also had a thin nose due to a hypoplastic alae nasi. METHODS The only member of the family who was not examined was individual I:1, described by relatives as having Twelve members of a family with ODDD from Cascavel, Bra- clinical characteristics typical of ODDD, who died at 63 zil, underwent screening for mutations in the GJA1 gene and a years of age, blind due to glaucoma. The Table gives the comprehensive ophthalmic examination. We defined ODDD ocular findings of all family members. Among the 8 af- on the basis of clinical characteristics described in the litera- fected individuals who were examined, all had micro- ture. Informed consent was obtained from the members of the family. Ophthalmic examination included intraocular pres- cornea (corneal diameters, 8-9 mm) and peripupillary iris sure measurement by means of applanation tonometry; slit- atrophy (Figure 3). Three (40%) of the affected indi- lamp biomicroscopy and gonioscopy; evaluation of the optic viduals, all of them belonging to the second generation, disc with a 78-diopter lens; automated perimetry (Humphrey had glaucoma. One of these was found to have open- System 24.2; Zeiss-Humphrey-Zeiss Systems, Dublin, Calif) in angle glaucoma and had undergone trabeculectomy individuals older than 15 years; measurements of the axial length in both eyes. In this individual, results of gonioscopy and corneal diameters and keratometry; and ocular history ob- disclosed a wide open angle, allowing the visualization tained by interview, including the age at diagnosis of glau- of the ciliary band. The other 2 family members had coma and clinical and surgical treatment. angle-closure glaucoma. Both had shallow anterior cham- Glaucoma was defined as the presence of at least 2 of the bers, and gonioscopy allowed the visualization of the following characteristics: (1) intraocular pressure above 24 mm Hg; (2) optic disc changes, including thinning of the neu- Schwalbe line. Results of indentation gonioscopy did not roretinal rim, hemorrhage, notch, cup-disc ratio greater than disclose peripheral anterior synechiae. 0.7, or asymmetry of cup-disc ratio greater than 0.2; and (3) The screening for mutations in the GJA1 gene iden- glaucomatous visual field defect, defined as a corrected pat- tified a new mutation at codon 59, changing a proline tern standard deviation outside the 95% normal limits or a glau- (CCT) for a histidine (CAT) in heterozygosis (P59H) coma hemifield test result outside the 99% limits. (Figure 4). This alteration segregated in members with We extracted DNA from peripheral blood and performed the ODDD phenotype (Figure 1). Results of analysis by direct sequencing of the GJA1 gene using fluorescent dideoxy- means of restriction fragment length polymorphism (MvaI nucleotides on an automated sequencer (ABI 310; Applied Bio- 15 enzyme) in the control group did not disclose this struc- systems, Foster City, Calif), according to Paznekas et al. Con- tural alteration. nexin amino-acid alignment was performed with the ClustalW program (http://www.ebi.ac.uk/clustalw/). Sequences were obtained from the Entrez-Protein Web site of the National Cen- COMMENT ter for Biotechnology Information (http://www.ncbi.nlm.nih .gov/entrez/). A control group of 60 healthy individuals This study described a Brazilian family with ODDD that underwent evaluation by means of digestion of the GJA1 gene carries a new mutation in the GJA1 gene. Among the ocu- PCR product with the MvaI enzyme. lar findings, open-angle and angle-closure glaucoma were observed in members of the second generation. RESULTS In a review by Loddenkemper et al4 that included 243 individuals with ODDD syndrome, 9 had the diagnosis This family included 13 subjects, with 9 affected indi- of glaucoma, 15 showed reduced visual acuity, and an- viduals distributed among 3 generations, suggesting an other 20 were described with other ocular manifesta-

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Figure 2. Phenotype of oculodentodigital dsyplasia in the studied family showing syndactyly involving the second and third toes (A) and mild clinodactyly (B) in family members II:2, III:1, and III:2 (depicted in Figure 1).

Table. Ophthalmic Characteristics of the Family With ODDD

Visual IOP, Results of Gonioscopy, C/D Vertical ∅ Cornea, Axial Individuals Acuity* Refraction, SE* mm Hg* Biomicroscopy Angle* Ratio* mm* Length, mm* KT, D* I:2 25/25 0.00/0.00 14/16 Normal Open/Open 0.4/0.4 12.0/12.0 22.15/22.16 43.25/43.50 II:1 25/20 ϩ2.50/ϩ2.50 14/14 Normal Open/Open 0.4/0.4 12.2/12.3 23.60/23.53 43.50/43.25 II:2†‡ 20/25 ϩ2.00/ϩ2.25 14/26 Peripupillary Narrow/Narrow 0.3/0.3 8.2/8.4 19.31/19.87 46.62/47.37 iris atrophy II:3†‡ 30/Ͻ400 −0.75/NA 30/30 Peripupillary Open/Open 0.4/0.5 8.5/8.2 21.53/23.63 47.50/48.12 iris atrophy II:4 20/20 −1.25/−0.75 12/12 Normal Open/Open 0.4/0.4 12.2/12.3 23.02/22.94 44.25/43.75 II:5 20/20 0.00/0.00 16/16 Normal Open/Open 0.3/0.3 12.0/12.0 22.95/23.20 46.00/45.50 II:6†‡ 40/20 ϩ1.75/ϩ0.75 25/14 Peripupillary Narrow/Narrow 0.7/0.6 8.0/8.0 20.87/20.79 45.37/45.25 iris atrophy III:1† NA NA 20/18 Peripupillary Narrow/Narrow 0.3/0.4 8.8/8.8 21.78/21.86 41.75/41.25 iris atrophy III:2† 20/25 0.00/0.00 17/20 Peripupillary Narrow/Narrow 0.3/0.3 8.4/8.2 21.22/21.14 44.62/44.75 iris atrophy III:3† 20/20 ϩ1.75/ϩ1.50 19/19 Peripupillary Open/Open 0.6/0.7 9.0/9.0 20.62/20.78 47.50/47.37 iris atrophy III:4† 20/20 0.00/−0.25 14/15 Peripupillary Open/Open 0.5/0.6 8.5/8.5 20.80/20.73 48.12/48.62 iris atrophy III:5† 30/20 ϩ1.00/ϩ0.25 16/16 Peripupillary Open/Open 0.3/0.3 8.0/8.0 20.87/20.85 45.37/45.37 iris atrophy

Abbreviations: C/D, cup-disc; D, diopter; IOP, intraocular pressure; KT, keratometry; NA, not available; ODDD, oculodentodigital dysplasia; SE, spherical equivalent; ∅, diameter. *Data presented as result in the right eye/result in the left eye. †Indicates family members with ODDD syndrome. ‡Indicates family members with glaucoma.

tions. However, that article emphasized the neurologi- tients with angle-closure glaucoma were described by cal aspects of ODDD. In another review by Judisch et al,9 Sugar et al12,13 in 1966 and 1978. the authors identified 6 patients with glaucoma, among In our study, among the 3 family members with glau- whom 2 had open-angle glaucoma and the remaining pa- coma, 1 had open-angle glaucoma and 2 had angle- tients had goniodysgenesis. Novotny and Sterbova10 de- closure glaucoma. When we consider the individuals in scribed 102 members of a 5-generation family, in which the third generation, all 5 family members with ODDD 42 were found to have ODDD. The condition was com- were young (age range, 8-20 years), and glaucoma had plicated by glaucoma in the older individuals and stra- not developed (Table). It will be interesting to follow up bismus in the younger ones. In 1996, Braun et al11 de- the association between glaucoma and ODDD among this scribed a boy with ODDD and juvenile open-angle generation of the family. It is highly possible that some glaucoma who showed reduced corneal diameter (OD, of them will develop glaucoma later in life. 9.0ϫ7.0 mm; OS, 8.5ϫ6.5 mm) with axial lengths close In2003,Paznekasetal15 described17familieswithODDD to the normal range (OD, 25.3 mm; OS, 23.6 mm). Tra- and 17 different structural alterations in the GJA1 gene. The boulsi and Parks8 described 2 individuals with ODDD as- mutations segregated with the disease and were absent in sociated with developmental glaucoma that started at 4 a control group of 100 individuals. The GJA1 gene is respon- months and 5 years of age, respectively. Finally, 2 pa- sible for the synthesis of the Cx43 protein. Connexins are

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D E

Figure 3. Presence of peripupillary iris atrophy in family members II:2 (with oculodentodigital dysplasia [ODDD] syndrome and angle-closure glaucoma [A]), II:6 (with ODDD syndrome and angle-closure glaucoma [B]), III:1 (with ODDD syndrome [C]), III:2 (with ODDD syndrome [D]), and III:5 (with ODDD syndrome [E]).

A C A C T C A G C A A C N T G G T T G T G A A A A T

B C A C T C A G C A A C C T G G T T G T G A A A A T

Figure 4. Direct sequencing of the sense strand of the polymerase chain reaction product of the gap junction protein alpha 1 (GJA1) gene from a family member with oculodentodigital dysplasia, showing a novel heterozygous missense mutation (A). The mutation, CCT→CAT, results in a change in amino acid at codon 59 (proline→histidine) that is absent in the control subject (B).

molecules that are structurally important for the formation turbances of several biological processes such as cardiac con- of gap junctions between adjacent cells, through which di- duction, auditory function, aging and senescence, neuro- rect intercellular communication via diffusion of ions and nalfunction,pathfindingandglialsignaling,immunesystem metabolites can occur.16 Several human diseases are asso- activation, bone and tooth development, neural tube de- ciated with mutations in connexin genes. These include dis- fects, hematopoiesis, and myogenesis.17 For example, con-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 genital cataract has been described as a result of Cx46 and in the trabecular meshwork during angle development Cx50 mutations.18,19 Both connexins are expressed during and in the trabecular meshwork of patients with glau- lens development, and their function seems to be depen- coma. dent on the repertoire, rather than the number, of these 2 connexin subunits available.20 In their study, Paznekas et al15 described 3 families with Submitted for Publication: June 16, 2004; final revision ODDD and glaucoma showing mutations located in the received December 5, 2004; accepted January 27, 2005. cytoplasm (Y17S) and in the first transmembrane do- Correspondence: José P. C. Vasconcellos, MD, PhD, Uni- main (G22E) and second transmembrane domain (L90V) versidade Estadual de Campinas–Disciplina de Oftalmo- of the GJA1 gene. The Y17S mutation was associated with logia, R: Tessália Vieira de Camargo, 126, Cidade Uni- early cataract development, and the G22E mutation was versitária Zeferino Vaz, Distrito de Barão Geraldo, Caixa identified in 2 children with developmental glaucoma. Postal 6111, CEP: 13083-970 ([email protected]). In the family described herein, we identified the P59H Financial Disclosure: None. mutation, which belongs to the first extracellular loop Funding/Support: This study was supported by grants 98/ of the connexin protein and has not been reported in any 13830-0 and 02/11575-0 from the Fundação de Amparo of the previously described 17 families. The extracellu- à Pesquisa do Estado de São Paulo, São Paulo, Brazil. lar loops of the connexin proteins are important for the docking of gap junctional hemichannels (connexons) and REFERENCES are responsible for selective compatibility between different types of connexins that lead to the formation of 1. Shapiro RE, Griffin JW, Stine OC. Evidence for genetic anticipation in the oculodentodigital 20,21 syndrome. Am J Med Genet. 1997;71:36-41. heterotypic functional channels. The proline resi- 2. Boyadjiev SA, Jabs EW, LaBuda M, et al. Linkage analysis narrows the critical region for ocu- due at codon 59 is close to the absolutely conserved cys- lodentodigital dysplasia to chromosome 6q22-q23. Genomics. 1999;58:34-40. 3. Lohmann W. Beitrag zur Kenntnis des reinen Mikrophthalmus. Arch Augenheilk. 1920;86:136- tein residues that are crucial for intramolecular stabili- 141. zation.22 Furthermore, this proline has been shown to be 4. Loddenkemper T, Grote K, Evers S, Oelerich M, Stogbauer F. Neurological manifestations of the oculodentodigital dysplasia syndrome. J Neurol. 2002;249:584-595. a highly conserved residue among the connexins of other 5. Meyer-Schwickerath G, Gruterich E, Weyers H. Mikrophthalmssyndrome. Klin Monatsbl Augenheilkd. 1957;131:18-30. species and among different human connexins. 6. 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