A consanguineous Arab pedigree in which recessive amelogenesis imperfecta (AI) and cone-rod dystrophy cosegregate, was screened for linkage to known retinal dystrophy and tooth abnormality loci by genotyping neighbouring microsatellite markers. This analysis resulted in linkage with a maximum lod score of 7.03 to the marker D2S2187 at the achromatopsia locus on chromosome 2q11, and haplotype analysis placed the gene(s) involved in a 2 cM/5 Mb interval between markers D2S2209 and D2S373. The CNGA3 gene, known to be involved in achromatopsia, lies in this interval but thorough analysis of its coding sequence revealed no mutation. Furthermore, affected individuals in four consanguineous recessive pedigrees with AI but without CRD were heterozygous at this locus, excluding it as a common cause of non-syndromic recessive AI. It remains to be established whether this pedigree is segregating two closely linked mutations causing disparate phenotypes or whether a single defect is causing pathology in both teeth and eyes.
Amelogenesis imperfecta (AI) is the collective term for an inherited group of dental diseases where the common clinical feature is an abnormality of tooth enamel. The enamel may be thin but otherwise normal (hypoplastic), or poorly mineralised (hypomineralised), or a combination of the two. AI can be inherited as an autosomal dominant, autosomal recessive or X-linked trait.1,2 Mutations have been identified in the amelogenin gene, AMGX (locus designated AIH1), on Xp22.1-22.3 in X-linked pedigrees.3 There is also a second locus on the X chromosome at Xq22-28 (AIH3).2,4 The only known locus for autosomal dominant AI to date (AIH2) is on chromosome 4q5 and the gene mutated in AI at this locus, enamelin, has recently been identified.6 Prior to this study, no locus had been reported for autosomal recessive AI, though pedigrees suggesting its existence have been documented.7,8 This mode of inheritance is particularly prevalent in Sweden, where autosomal recessive families are thought to account for 12% of cases in one study.7 Syndromic forms of AI can be associated with: epilepsy (MIM: 226750), platyspondyly (MIM: 601216), nephrocalcinosis (MIM: 204690) sensorineural hearing loss (MIM: 234580) cone-rod dystrophy (MIM: 217080) in the pedigree studied here and with keratin and bone abnormalities in tricho-dento-osseous syndrome (MIM: 190320).
Cone-rod dystrophies (CORD) are a group of retinal disorders characterised by degeneration of cone and subsequently rod photoreceptors. Clinically this is manifest as an initial loss of central vision, colour vision and photophobia with subsequent night blindness and restricted visual fields as rod involvement begins.9 Several genes have been implicated in autosomal dominant cone-rod dystrophy; namely peripherin/RDS,10 CRX,11 RETGC-112 and GUCA1A.13 A further locus exists; CORD7 on 6q11-q1514 for which the gene has yet to be identified. Loci for recessively inherited forms of cone-rod dystrophy are less well characterised. Four loci exist; these are CORD5 on 17p13-p12,15 CORD 8 on 1q12-q24,16 CORD9 on 8p12-q1117 and the ABCA4 gene on 1p21-p22.18
In this study we describe linkage analysis in a large consanguineous autosomal recessive Arab pedigree originating from the Gaza strip in which all 29 affected family members studied were found to suffer from both cone-rod dystrophy and amelogenesis imperfecta (Figure 1). Their phenotype is described in detail elsewhere19 and their visual complaints can be summarised as colour blindness, photophobia, nystagmus and poor visual acuity (beginning within the first 2 years of life). Older patients begin to show loss of the scotopic ERG, reduction in peripheral visual fields and optic disc pallor, characteristic of rod photoreceptor involvement. Fundus examination revealed an early bulls eye maculopathy with later chorioretinal atrophy at the macula producing a macular coloboma. AI was found to co-segregate perfectly with the retinal dystrophy; enamel was either absent or vestigial on erupted and unerupted teeth. Three teeth were examined microscopically and showed a generalised absence of enamel with scanty areas of full thickness enamel away from the occlusal surface.19
Materials and Methods
Genomic DNA was extracted from peripheral blood leukocytes using standard techniques from 21 affected and 16 unaffected family members. Initial analysis was performed on pooled equimolar DNA solutions from affected individuals, and results were compared with those obtained in pooled carriers and unaffected siblings. These DNA mixes were genotyped by PCR using standard parameters and incorporating 32P-labelled dCTP. Fragments were resolved on 50 cm denaturing polyacrylamide gels, then visualised by autoradiography. After linkage had been established, individual fluorescently tagged polymorphic microsatellite markers from the region were amplified by PCR using genomic DNA from a cohort of 34 family members (Figure 1) and the products were resolved using an ABI 377 automated DNA sequencer (Applied Biosystems). Products were identified and sized using GENESCAN (version 2.0.2) and GENOTYPER (version 1.1.1) software. A Lod score was obtained using the CYRILLIC 2.1 data management package and the MLINK program from the LINKAGE package (VERSION 5.1) software.20 Penetrance was assumed to be 100%.
Exons of the CNGA3 gene were screened in this pedigree by direct sequencing of both DNA strands in two affected family members. Primers for sequencing were designed to include a minimum of 20bp of intronic sequence flanking each exon. Exons were PCR amplified, purified using the Qiaquick PCR purification kit (Qiagen) then sequenced directly using the ABI Prism BigDye Terminator cycle sequencing kit from Applied Biosystems.
DNAs consisting of pooled affected individuals and carriers/normal siblings were screened for linkage using 121 polymorphic microsatellite markers closely linked to known retinal dystrophy loci and a further three markers at the known autosomal AI locus (AIH2). The retinal dystrophy loci tested were those reported up to 1996, and included seven dominant and seven recessive RP loci, eight central retinal dystrophy loci, five for Ushers syndrome, four for Bardet Biedl syndrome and eight other loci at which mutations cause an assortment of retinal phenotypes. Subsequently a locus on chromosome 2q11 was implicated in achromatopsia, another retinal disease.21 We noted the clinical similarities between the retinal phenotype in the Gaza pedigree studied herein and this disease. Achromatopsia (total colour blindness; MIM: 216900) is an autosomal recessive retinal disorder in which patients are unable to differentiate colours. They are photophobic and have a markedly reduced level of daytime visual acuity from early childhood together with nystagmus. All of these features are prominent in the phenotype studied here. In addition however, members of this pedigree show progressive retinal degeneration with involvement of both rods and cones. The marker D2S417 on chromosome 2q, known to be closely linked to the achromatopsia locus, was therefore tested on pooled DNAs from affected and normal individuals, and this analysis revealed excess allele sharing.
We genotyped further markers spanning the achromatopsia locus on 2q11 (ACHM2) in a panel of individual family members. Polymorphic microsatellite markers D2S417, D2S388, D2S2175, D2S2209, D2S2311, D2S2187, D2S1309, D2S373 D2S436 and D2S340 were amplified by PCR using genomic DNA from a cohort of 34 family members. A maximum Lod score of 7.03 was obtained for marker D2S2187. Genotyping of affected individuals revealed a region of homozygosity of approximately 2 cM (5 Mb) flanked by markers D2S2209 and D2S373, as shown in Figure 1. This result is the first report of a locus for autosomal recessive AI and is also confirmation of a further locus for autosomal recessive CORD.
Additional genotyping was performed for markers D2S2187 and D2S1309 on four other consanguineous recessive pedigrees in which AI was segregating in the absence of CORD. No homozygosity was seen for any pedigree with either marker. It is therefore unlikely that AI in any of these pedigrees results from mutations at this locus.
In view of the clinical similarities between achromatopsia and CORD in this pedigree, and in the light of a recent report of CNGA3 mutations in patients with progressive central retinal dystrophies22, CNGA3 appeared a strong candidate gene for a mutation causing the retinal phenotype in this pedigree. CNGA3 encodes the alpha subunit of a cyclic GMP-gated channel present in the plasma membrane of the cone-photoreceptor outer segment. This cyclic GMP-gated channel is responsible for the influx of sodium and calcium ions as a response to light and represents a key element of the phototransduction cascade in cone photoreceptors.23 The genomic sequence of CNGA3 is contained within the genomic clone AC027241 along with marker D2S2311 (Goldenpath), one of the markers contained in the region of homozygosity in our pedigree, providing good evidence for its consideration as a positional candidate. All seven exons of this gene, together with the two new exons (Exons 0 and 2b) described recently22 were screened by direct sequencing of both DNA strands in two affected family members. A polymorphism was found in the 5′UTR which appeared to segregate with disease (84014A>T, reference sequence AC092675). The existence of a SNP at this site had been previously described by B Wissinger et al (personal communication). In addition two new variants were found within the 5′UTR although neither segregated with disease.
In order to further ensure the accuracy of the sequencing data, the entire CNGA3 gene was then rescreened independently using direct sequencing in a second lab (Tuebingen). This analysis confirmed the absence of causal mutations anywhere within the coding sequence or within the 5′ region surrounding exon 0, including a putative promoter site, located approximately 24 kb upstream of the start codon.
These data demonstrate the existence of a locus for autosomal recessive AI and CORD on chromosome 2q11. In addition this study reveals locus heterogeneity in autosomal recessive AI, since this locus is excluded in four smaller consanguineous AI pedigrees. However two questions remain. Firstly, could the CNGA3 gene still be involved in the retinal phenotype seen in this pedigree? Though mutations in the coding sequence, splice sites and putative promoter have been excluded, an intronic change or large-scale rearrangement within the gene or in nearby sequence remains a possibility. Also large-scale genomic changes up to 1 Mb away from the gene in question have been implicated in inherited human diseases, altering the level of transcription of the gene with a so-called position effect.24 Such an effect might alter the expression of several genes in the vicinity and could therefore account for both tooth and retinal phenotypes. An alternative hypothesis, that mutations in a second gene within this relatively small interval could result in such a similar retinal phenotype to that described for CNGA3 mutations seems unlikely. A similar hypothesis was proposed to explain the lack of mutations in the RPGR gene in RP3 linked, retinitis pigmentosa families.25 This was subsequently disproved with the discovery of a mutational hotspot in a previously unidentified RPGR exon.26
Secondly, it remains to be determined whether the two disparate phenotypes in this pedigree result from a defect in a single gene or from defects in two closely linked genes. In the light of this question it is interesting to note that expression of CNGA3 is not confined to cone photoreceptors, with evidence of expression in bovine kidney, spermatozoa, heart and colon.27 Identification of genes expressed during enamel formation is far from complete and so it is a matter of speculation as to whether expression of CNGA3 occurs. There is however evidence for transcellular active transport of calcium through ameloblasts, the cells involved in the mineralisation of enamel.28,29,30
A further 23 characterised or predicted genes map within the interval predicted to contain the mutation(s) in this pedigree (Goldenpath) defined by haplotype analysis. Of these, none are retina specific, four are hypothetical proteins with no known function and four others are ubiquitously expressed, including expression in brain and/or eye. No genes are documented as having expression in teeth. It remains possible that these could be involved in one or both of the phenotypes seen in this family.
Analysis of the contig available for this region using the human genome browser (UCSC: Goldenpath) shows a discrepancy for the position of the marker D2S2209. Haplotype reconstruction in this pedigree reveals a proximal crossover in this marker, which correlates well with its position on the Marshfield genetic map where it is placed proximal to D2S2175 but distal to D2S2311. Its position on the contig is however distal to D2S1309. This marker order depends upon the arrangement of clones bridging a gap in genomic sequence, which are linked together by the gene LAF4. Until the finished draft of the sequence is available it will be impossible to be certain of the true marker order for the region. However if the Goldenpath marker order were confirmed, the locus would be further refined to a 2Mb interval between markers D2S2209 and D2S373, and could potentially exclude CNGA3 from the disease interval.
In summary, we have identified a new locus on chromosome 2q11 at which recessive CORD and AI cosegregate. Further experiments will be required to determine whether this represents two distinct but closely linked phenotypes or a new syndromic locus where both CORD and AI result from mutations in the same gene. The CNGA3 gene remains a strong candidate for involvement in the retinal phenotype although it has been sequenced in its entirety in this pedigree and no mutations were identified.
Witkop Jr CJ . Amelogenesis imperfecta, dentinogenesis imperfecta and dentin dysplasia revisited: problems in classification J Oral Pathol 1988 17: 547–553
Aldred MJ, Crawford PJ, Roberts E et al. Genetic heterogeneity in X-linked amelogenesis imperfecta Genomics 1992 14: 567–573
Lagerstrom M, Dahl N, Nakahori Y et al. A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIH1) Genomics 1991 10: 971–975
Crawford PJ, Aldred MJ . Clinical features of a family with X-linked amelogenesis imperfecta mapping to a new locus (AIH3) on the long arm of the X chromosome Oral Surg Oral Med Oral Pathol 1993 76: 187–191
Karrman C, Backman B, Dixon M, Holmgren G, Forsman K . Mapping of the locus for autosomal dominant amelogenesis imperfecta (AIH2) to a 4-Mb YAC contig on chromosome 4q11-q21 Genomics 1997 39: 164–170
Rajpar MH, Harley K, Laing C, Davies RM, Dixon MJ . Mutation of the gene encoding the enamel-specific protein, enamelin, causes autosomal-dominant amelogenesis imperfecta Hum Mol Genet 2001 10: 1673–1677
Backman B, Holmgren G . Amelogenesis imperfecta: a genetic study Hum Hered 1988 38: 189–206
Chosack A, Eidelman E, Wisotski I, Cohen T . Amelogenesis imperfecta among Israeli Jews and the description of a new type of local hypoplastic autosomal recessive amelogenesis imperfecta Oral Surg Oral Med Oral Pathol 1979 47: 148–156
Moore AT . Cone and cone-rod dystrophies J Med Genet 1992 29: 289–290
Bascom RA, Liu L, Heckenlively JR, Stone EM, McInnes RR . Mutation analysis of the ROM1 gene in retinitis pigmentosa Hum Mol Genet 1995 4: 1895–1902
Freund CL, Gregory-Evans CY, Furukawa T et al. Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor Cell 1997 91: 543–553
Kelsell RE, Gregory-Evans K, Payne AM et al. Mutations in the retinal guanylate cyclase (RETGC-1) gene in dominant cone-rod dystrophy Hum Mol Genet 1998 7: 1179–1184
Payne AM, Downes SM, Bessant DA et al. A mutation in guanylate cyclase activator 1A (GUCA1A) in an autosomal dominant cone dystrophy pedigree mapping to a new locus on chromosome 6p21.1 Hum Mol Genet 1998 7: 273–277
Kelsell RE, Gregory-Evans K, Gregory-Evans CY et al. Localization of a gene (CORD7) for a dominant cone-rod dystrophy to chromosome 6q Am J Hum Genet 1998 63: 274–279
Balciuniene J, Johansson K, Sandgren O, Wachtmeister L, Holmgren G, Forsman K . A gene for autosomal dominant progressive cone dystrophy (CORD5) maps to chromosome 17p12-p13 Genomics 1995 30: 281–286
Khaliq S, Hameed A, Ismail M et al. Novel locus for autosomal recessive cone-rod dystrophy CORD8 mapping to chromosome 1q12-Q24 Invest Ophthalmol Vis Sci 2000 41: 3709–3712
Danciger M, Hendrickson J, Lyon J et al. CORD9 a new locus for arCRD: mapping to 8p11, estimation of frequency, evaluation of a candidate gene Invest Ophthalmol Vis Sci 2001 42: 2458–2465
Cremers FP, van de Pol DJ, van Driel M et al. Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR Hum Mol Genet 1998 7: 355–362
Jalili IK, Smith NJ . A progressive cone-rod dystrophy and amelogenesis imperfecta: a new syndrome J Med Genet 1988 25: 738–740
Lathrop GM, Lalouel JM, Julier C, Ott J . Strategies for multilocus linkage analysis in humans Proc Natl Acad Sci USA 1984 81: 3443–3446
Kohl S, Marx T, Giddings I et al. Total colourblindness is caused by mutations in the gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated cation channel Nat Genet 1998 19: 257–259
Wissinger B, Gamer D, Jagle H et al. CNGA3 mutations in hereditary cone photoreceptor disorders Am J Hum Genet 2001 69: 722–737
Bonigk W, Altenhofen W, Muller F et al. Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels Neuron 1993 10: 865–877
Bedell MA, Jenkins NA, Copeland NG . Good genes in bad neighbourhoods Nat Genet 1996 12: 229–232
Fujita R, Buraczynska M, Gieser L et al. Analysis of the RPGR gene in 11 pedigrees with the retinitis pigmentosa type 3 genotype: paucity of mutations in the coding region but splice defects in two families Am J Hum Genet 1997 61: 571–580
Vervoort R, Lennon A, Bird AC et al. Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa Nat Genet 2000 25: 462–466
Kaupp UB . Family of cyclic nucleotide gated ion channels Curr Opin Neurobiol 1995 5: 434–442
Bawden JW . Calcium transport during mineralization Anat Rec 1989 224: 226–233
Vicars TM, Stanfield CN, Crenshaw MA, Bawden JW . The effects of ATP depletion and ionophore A23187 on calcium transport in the secretory rat enamel organ Arch Oral Biol 1983 28: 513–516
Kawamoto T, Shimizu M . Pathway and speed of calcium movement from blood to mineralizing enamel J Histochem Cytochem 1997 45: 213–230
We would like to express our thanks to the Wellcome Trust for funding this research (grants 055145/Z/98 and 035535/Z/96)
About this article
Cite this article
Downey, L., Keen, T., Jalili, I. et al. Identification of a locus on chromosome 2q11 at which recessive amelogenesis imperfecta and cone-rod dystrophy cosegregate. Eur J Hum Genet 10, 865–869 (2002). https://doi.org/10.1038/sj.ejhg.5200884
- amelogenesis imperfecta
- cone-rod dystrophy
- retinal degeneration
Journal of Medical Genetics (2019)
Jalili Syndrome: Cross-sectional and Longitudinal Features of Seven Patients With Cone-Rod Dystrophy and Amelogenesis Imperfecta
American Journal of Ophthalmology (2018)
Ophthalmic Genetics (2017)
European Journal of Medical Genetics (2017)
Characterization of the nanoscratch, microstructure, and composition in hypoplastic amelogenesis imperfecta
Advances in Mechanical Engineering (2015)