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The genetic basis of non-syndromic autosomal recessive forms of amelogenesis imperfecta (AI) is unknown. To evaluate five candidate genes for an aetiological role in AI. In this study 20 consanguineous families with AI were identified in whom probands suggested autosomal recessive transmission. Family members were genotyped for genetic markers spanning five candidate genes: AMBN and ENAM (4q 13.3), TUFT1 (1q21), MMP20 (11q22.3-q23), and KLK4 (19q13). Genotype data were evaluated to identify homozygosity in affected individuals. Mutational analysis was by genomic sequencing. Homozygosity linkage studies were consistent for localisation of an AI locus in three families to the chromosome 4q region containing the ENAM gene. ENAM sequence analysis in families identified a 2 bp insertion mutation that introduced a premature stop codon in exon 10. All three probands were homozygous for the same g.13185_13186insAG mutation. These probands presented with a generalised hypoplastic AI phenotype and a class II openbite malocclusion. All heterozygous carriers of the g.13185_13186insAG mutation had localised hypoplastic enamel pitting defects, but none had AI or openbite. The phenotype associated with the g.13185_13186insAG ENAM mutation is dose dependent such that ARAI with openbite malocclusion segregates as a recessive trait, and enamel pitting as a dominant trait.
J Med Genet 2003;40:900-906
The amelogenesis imperfectas (AI) are a clinically and aetiologically heterogeneous group of heritable disorders characterised by qualitative or quantitative anomalies of enamel development. While syndromic and non-syndromic forms of AI are reported, non-syndromic forms are the most prevalent. (1) (2) Although multiple classifications systems have been proposed for this condition, our current understanding of its aetiology does not allow the development of a robust nosology to account for the phenotypic variability observed. (3-5)
Both X linked and autosomal transmission of non-syndromic forms of AI are well documented. (6-9) Most forms of X linked AI are caused by mutation of the amelogenin gene, (AMELX (10) ). (11) The most common autosomal forms of AI show dominant transmission. Genetic linkage studies indicate that at least two distinct autosomal loci are responsible for dominant forms of AI, with some cases caused by enamelin (ENAM (12) ) gene mutations. (5) (13-17) Identification of genes responsible for X linked and autosomal dominant forms of AI has contributed to a better understanding of the disease pathogenesis, and in some cases, genotype-phenotype correlations are emerging. (18)
In contrast to the success in identifying the genetic basis of autosomal dominant and X linked forms of AI, genetic studies have not identified or localised genetic loci for non-syndromic forms of autosomal recessive AI (ARAI). Based on biological function and tissue expression, five candidate genes have been proposed for autosomal forms of AI, including ameloblastin (AMBN), enamelin (ENAM), tuftelin (TUFT1), enamelysin (MMP20), and kallikrein 4 (KLK4). (14) (19-24) To evaluate support for or against linkage of these candidate loci with non-syndromic ARAI, we undertook homozygosity linkage studies in 20 nuclear families. In this paper we report identification of a novel ENAM mutation in probands from three families. Homozygous carriers of this novel mutation show AI and openbite malocclusion, while heterozygous carriers have only a mild localised enamel pitting phenotype.
Pedigrees and diagnosis
Affected individuals were identified by proband ascertainment from dental clinics at the School of Dentistry, University of Istanbul, Istanbul, Turkey in accordance with institutional review board approval from the University of Istanbul and the University of Pittsburgh. Available family members had an oral examination and dental radiographs to characterise the AI phenotype when present. The presence of the disorder was established by generalised yellow-brown discolouration of the teeth, decreased enamel mineralisation, and pathological loss of enamel. Using modifications of clinical criteria previously proposed by Witkop, (1) affected individuals were classified into one of four groups: hypoplastic; hypomaturation; hypocalcified; and hypomaturation/hypoplasia with taurodontism. (3)
In addition to oral examinations, complete medical histories were taken to identify any additional clinical findings that would be consistent with syndromic conditions. Individuals with syndromic presentations of AI were excluded from the analyses.
Cephalometric and panoramic radiographs, photographic records, and dental casts were obtained to characterise the presence of skeletal openbite malocclusion in individuals with clinically evident openbite. Standard lateral cephalography was undertaken using a Cranex-3+Ceph at 10 mA and 81 kV (Cranex Co, Helsinki, Finland). Conventional cephalometric landmarks were identified and 15 angular and 15 linear measurements were made. (25) Negative values for the recordings of overbite represent a dentoalveolar anterior openbite. The facial ratio expressed by the relation between upper face height and total face height was calculated from the formula N-ANS/N-Me x 100, where N-ANS = mid facial height and N-Me = total face height.
Molecular genetic analyses
DNA marker analysis
Genomic DNA was isolated from whole blood by standard techniques using the QIAamp blood kit (Qiagen). Family members were genotyped for STRP (short tandem repeat polymorphism) genetic markers spanning the five autosomal candidate loci, selected on the basis of their role in enamel development. Three gene loci (TUFT1, MMP20, and KLK4) were genotyped using previously described STRPs. (17) Although the AMBL and ENAM loci have been mapped to chromosome 4q21, their precise location and position relative to each other has only recently been determined. (17) Three novel STRP loci spanning the AMBL and ENAM loci were identified using the tandem repeats finder program. (26) Oligonucleotide primers used to amplify these loci were: 719M16: F 5'-CCATTAGAGCTATTTCAGATA-3'; R 5' - CACAAAGCCAGAGAA ACATTA- 3'; AMB: F 5'-GATTCAGCATCAACTTCCACAGAG-3': R 5'-CCAGCCTGAGCTTGTCTTAAAGAT-3'; and 92H22: F 5'-CTCGCTTTGGGTTTCTCTGATGTT-3', R 5'- CTGGCA ATGTGGGCTGRCTCTACA-3'.
Marker loci were amplified by polymerase chain reaction, using fluorescence labelled primers, permitting genotyping by conventional …