Diversity of clinical, radiographic and genealogical findings in 41 families with amelogenesis imperfecta

Abstract Amelogenesis imperfecta (AI) is a group of enamel development disorders that alter the structure and chemical composition of the tissue. There is great variability in the clinical presentation; according to Witkop, AI can be categorized into 14 subtypes, which makes its diagnosis extremely complex. Objective: This study aimed to describe and determine the frequency of clinical and radiographic features and inheritance patterns found in 41 Chilean families diagnosed with diverse types of AI. Material and Methods: We analyzed the clinical records, photographs, pedigrees and radiographs of 121 individuals recruited between 2003 and 2016. All of the information was included in a database that was analyzed using the application Stata 14. Results: The 72 affected individuals had average age of 16 years, and no sex association with the presence of AI was found. The most frequent clinical subtypes were as follows: 43% hypomature, 25% hypoplastic, 21% hypomature/hypoplastic, 7% hypocalcified and 4% hypocalcified/hypoplastic. The number of severely affected teeth was 22, which occurred in the patients with hypocalcified and hypocalcified/hypoplasic AI who presented the highest number of damaged teeth. Caries and periodontal disease were found in 47 and 32% of the patients, respectively. Malocclusions were observed in 43% of the individuals with AI, with open bite being the most frequent. Radiographically, the thickness of the enamel decreased in 51% of the patients, and 80% showed decreased radiopacity of the enamel compared to that of dentin. Autosomal dominant inheritance pattern was found in 37% of the families with hypoplastic AI, and autosomal recessive pattern was present in 56% of the other clinical subtypes, but more frequently in those affected with hypomature and hypocalcified AI. Conclusion: Of the five clinical subtypes, autosomal recessive hypomature, autosomal dominant hypoplastic and autosomal recessive hypomature/hypoplastic AI were the most prevalent subtypes in this group.


Introduction
Normal enamel is synthesized during tooth development as an extracellular matrix in a process called amelogenesis, which occurs in two stages. In the secretory stage, the ameloblast produces a partially mineralized protein matrix, which will correspond to the adult enamel. In the maturation stage, the protein matrix is degraded, and mineralization is completed. 1,2 Amelogenesis imperfecta (AI) is a group of lowprevalence hereditary conditions that cause alterations in the structure and chemical composition of the enamel matrix during development. [1][2][3] Currently, the diagnosis of AI involves a clinical and radiographic examination, and when possible, morphological analysis, using ground sections and scanning electron microscopy of the teeth, and molecular genetic analysis of the DNA samples can be performed. [3][4][5] AI is classified into three main types that are related to the stages of the tissue formation process. 6 A fault in the secretory stage of amelogenesis produces the hypoplastic type of AI, which is characterized by enamel that is thinner than normal and that contrasts normally from dentine in the radiographic analysis. 7,8 In hypocalcified AI, there is an alteration in the initial mineralization of the secretory stage; the enamel initially develops normal thickness, is orange-yellow at eruption and consists of poorly calcified matrix that is rapidly lost during normal function. In addition, the enamel has a lower radiopacity than the dentin. 6,7 In hypomature AI, the defect occurs in the maturation stage of the enamel; is of normal thickness but has a mottled appearance; is slightly softer than normal enamel; and chips from the crown. Radiographically, it presents with approximately the same radiodensity as that of dentin. 6,9 To adequately evaluate AI, a differential diagnosis with other defects and an investigation to determine if the enamel alterations are linked to environmental disturbances or have a discernible genetic transmission pattern are necessary. 2,10,11 The signs frequently found in the enamel are a decrease in thickness, rough texture, mottled appearance, the presence of cavitations (pits), horizontal or vertical bands and a dull white, yellow or brown color. In addition, a delay in eruption, dental impaction, anterior or lateral open bite, ogival palate, gingival inflammation, teeth with a decreased coronary sector and multiple diastemas can be observed. [12][13][14] Because AI is a rare and heterogeneous condition from a clinical and genetic point of view, dentists, in general, have difficulty in making a correct diagnosis regarding the presence of AI and the determination of its clinical subtype. In our experience, many professionals who do not know about this condition and its consequences in long-term treatment choose to refer these patients, which contributes to a greater psychosocial impact on them because they feel marginalized and are often left without a specific treatment solution and prognosis of their condition. known to be a sign of systemic or syndromic conditions that could be associated with AI. In-depth interviews were conducted with at least two family members by a geneticist in order to construct their genealogies and analyze the inheritance pattern. Additionally, clinical photographs were taken, and complementary tests were requested such as periapical and panoramic radiographs.
The presence of alterations in the enamel was recorded in relation to the glossiness, texture, color, thickness and surface defects. The gloss of the enamel was considered abnormal when it had an opaque appearance. The texture was evaluated by gently passing the dental probe over the enamel, and the thickness of the enamel was considered diminished when there was presence of generalized diastemas, pits or bands; the evaluation was complemented by the radiographic image. A radiographic analysis was conducted to evaluate the radiopacity and thickness of the enamel as well as the presence of dental and bone alterations.
The diagnosis of AI and its respective subtypes was based on the criteria described in the classification of Witkop 6 (1989); however, we considered only the following three main types of AI and their possible combinations: "type I-Hypoplastic, type II-Hypomaturation and type III-Hypocalcified".
Taurodontism was recorded as one more characteristic, among several others, that are associated with AI and not as "type IV-Hypomaturation-hypoplastic with taurodontism" from the Witkop's classification.
All of the information collected was included in a single database, and the statistical analysis The significance was determined at a value of P<0.05.

Sample description
A total of 121 individuals were evaluated from 41 Chilean families with an AI diagnosis; 58% of the cases were female ( Table 1). Most of the evaluated patients presented clinically with AI (60%), and the remaining individuals were direct relatives who did not manifest the disease. Although the number of women was higher in the group of affected individuals, no sex association was found in relation to the presence of AI (P=0.320). The group of individuals affected with AI had a significantly lower median age than the unaffected group (P=0.0004) ( Table 1).

Genealogical analysis
A significantly higher number of families affected with AI (56%) presented an autosomal recessive inheritance pattern (P=0.006) ( of the patients presented some type of dental symptomatology, and sensitivity to thermal changes was the most frequently reported symptom (11%).
It is interesting to note that periodontal disease, the presence of diastemas, high or low lip frenum insertion and ogival palate occurred with frequencies equal or greater than 22% (Table 3).

General and specific clinical characteristics of enamel
With regard to glossiness and texture, 41% of the patients showed an opaque enamel, and 46% of them showed that the texture of the tissue was rough ( Table   4). The most commonly observed enamel coloration (65% of the patients) was white/opaque with absence of translucency, which was more frequent than other colorations (P<0.0001). Opaque white spots were observed in a high percentage of the patients with AI (77%). Regarding surface defects of the enamel, 29% of the individuals had pitting and 65% showed wear (Table 4).
Radiographic characteristics of the enamel Radiographic analysis (Table 5) showed that normal and decreased enamel thickness occurred equally in individuals with AI. A decrease in the radiopacity of the enamel compared to that of dentine was observed in 80% of the patients. In this sample, only 16% of the patients presented taurodontism.

Clinical analysis of phenotypes
Five clinical subtypes of AI were identified ( Figure   1). The most frequently observed clinical subtypes in the 72 affected patients were hypomature AI (43%), followed by hypoplastic AI (25%) and hypomature/ hypoplastic AI (21%). Clinical subtypes of hypocalcified AI and hypocalcified/hypoplastic AI were found in 7% and 4% of the patients, respectively.

Hypoplastic Amelogenesis Imperfecta (HPAI)
The 18 cases of HPAI presented enamel thickness alterations such as a generalized decrease in thickness, which occurred in 82% of the cases (Table 4, Figures   2A and 2B), cavitations or pits in 41% of the cases, and the presence of horizontal bands in 29% of the patients (Table 4). Changes in enamel thickness were not similar within the same family. Different degrees of affection were also observed in the same individual as shown in Figure 2B. Diastemas were observed in 56% of cases, and dentin sensitivity was observed in 33% of the patients (Table 3). Dental wear and exodontias were very frequent in this group, which were observed in 94% and 56% of the cases, respectively (Tables 3   and 4, Figures 2A and 2B). Other clinical alterations were opaque white spots in 53% of the patients, rough texture in 47% of the patients, and opaque enamel in 41% of cases (Table 4).
Alterations in occlusion were observed in 50% of the patients, with open bite being the most frequent finding (28% of the cases) ( Table 3). Of the 18 cases of HPAI, radiographic images were available in 11 cases with evident enamel thickness reduction in 91%, and radiopacity in 82% of the cases (Table 5 and Figure 2C). Of the 11 cases analyzed, none presented taurodontism (Table 5). Of the 6 families with HPAI, 83% presented an autosomal dominant pattern of inheritance (Table 2).

Hypomature Amelogenesis Imperfecta (HMAI)
Thirty-one cases were classified in this group, from which the most frequently observed enamel characteristics were opaque white spots in 100% of the   cases and normal texture in 66% of the cases (Table 4 and Figures 2D and 2E). It is important to highlight that periodontal disease and malocclusions affected 29% of the patients (Table 3). In the radiographic study, a normal enamel thickness was observed in 79% of the cases, and a decrease in radiopacity was observed in 18 cases (75%) (Table 5, Figure 2F)  autosomal recessive type (14 families, 74%) ( Table 2).

Hypocalcified Amelogenesis Imperfecta (HCAI)
The 5 cases of HCAI had opaque and rough enamel and decreased thickness (Table 4). In 60% of the cases, the enamel was brown, and in the remaining two cases, it was yellow (Table 4, Figures 2G and 2H).
Regarding other clinical characteristics, 80% of the cases presented malocclusions, which were mainly open bite, whereas 60% of the cases presented periodontal disease, and 40% presented sensitivity to both thermal and chemical stimuli ( Table 3). This clinical subtype presented more affected teeth than the other clinical subtypes. The imaging showed that all of the cases had less radiopacity of the enamel compared to dentin (Figure 2I), and 80% of the cases presented a decrease in the enamel thickness of the erupted teeth (Table 5). An interesting finding was that 40% of the patients presented taurodontism (Table   5). In this group, 80% of the families presented an autosomal recessive inheritance pattern (Table 2).

Hypomature/Hypoplastic Amelogenesis Imperfecta (HM/ HPAI)
In this group, both HMAI and HPAI characteristics were observed in the same patient ( Figures 2J and   2K). The characteristics of HMAI were as follows: opaque enamel in 27% of cases, rough enamel in 40% of the patients, white/opaque enamel in 73% of cases and opaque white spots in 93% of cases (  Table 3). The radiographic study showed a decrease in radiopacity in 75% of cases (Table 5 and Figure 2L), a generalized decrease in enamel thickness in 50% of cases, and taurodontism in 42% of cases (Table 5).
In this subtype of AI, 50% of the families showed an autosomal recessive inheritance pattern ( Table 2).

Hypocalcified/Hypoplastic Amelogenesis Imperfecta (HC/ HPAI)
This group was formed by 3 individuals with presence of restorations, sensitivity to thermal changes and diastemas (Table 3). These 3 patients presented rough opaque enamel and a generalized decrease in thickness (Table 4, Figures 2M and 2N).
The color of the enamel varied from brown to yellow and white-yellow, and 1 patient presented with pits or cavitations ( Table 4). The radiographic study showed a generalized decrease in enamel thickness and decreased radiopacity compared to dentin ( Table   5, Figure 2O). Of the 3 families analyzed, only 2 had autosomal dominant inheritance ( * Radiopacity of the enamel in relation to the dentin Table 5-Distribution of the radiographic characteristics observed in the individuals affected with Amelogenesis Imperfecta according to clinical subtype The majority of individuals with AI had exclusively permanent dentition and a higher frequency of tooth loss due to exodontias (39%), which highlights the need for early diagnosis and intervention in these patients. Because 47% of the patients with AI present caries, exodontias can be justified. The high frequency of caries is consistent with previous reports in which more caries were observed in patients with AI than in healthy individuals. 14,22,23 However, the number of teeth with caries in patients with AI in this study was similar to that found in healthy 12-year-old Chilean children 24 .
This suggests that caries in individuals with AI could be explained by the time required to find a specialist that can diagnose them and provide adequate treatment, in addition to the intrinsic susceptibility of their defective It is interesting to note that all individuals with hypocalcified AI, alone or associated with hypoplastic AI, presented enamel with opaque gloss, rough texture and reduced thickness. The majority of patients with hypomature and hypoplastic AI phenotypes presented whitish enamel. The decrease in thickness was frequently observed in the hypoplastic type of AI, but a normal enamel thickness was present in 90% of the patients with hypomature AI and 60% in the combined type of hypomature/hypoplastic AI.
Finally, regarding clinical analysis, it is important to point out that, as reported in the literature, 6,10,15,17 we also found great variability in the phenotypic expression of AI. We observed differences in the teeth of the same patient and between members of the same family.

Radiographic analysis
In the radiographic evaluation, a similar frequency of patients with normal and diminished enamel thickness was observed. However, a decrease in the radiopacity of the enamel when compared with dentin was an important feature for the majority of the patients with AI. Patients with hypoplastic AI showed more frequent decreased enamel thickness and radiopacity. Most of the patients with hypomature AI presented with enamel with normal thickness and diminished radiopacity compared to dentin, and individuals with hypocalcified AI showed a tendency to have decreased enamel thickness and radiopacity.
Although a 40% prevalence of taurodontism has been described in patients with AI, 6,13,30 in the present study, a low percentage of individuals with this characteristic was found.

Genealogical analysis
In this study, 23 families (56%) presented significantly more autosomal recessive inheritance patterns, which corroborates with Wright's study 10 In our study, the X-linked pattern of inheritance was absent. However, a classic article on AI that involved a significant number of families established that approximately 5-10% of all the cases of AI are X-linked. 15 In this context, Wright's work with 71 families reported that 6 (23%) cases presented X-linked patterns of inheritance. The study by Chan 17 (2011), carried out with 39 families, showed that 4 of them (12%) presented this type of inheritance.
Considering this information, in two of our families with hypomature/hypoplastic AI, there were two possible inheritance patterns involved, one of which was probably X-linked, but this was not possible to determine. This is due to a lack of information in the pedigree and to the fact that in these cases, it was Although the main strength of this 13-years-long study is the inclusion of a large number of patients with AI in its sampling, it also has some limitations related to our nonrandom convenience sample formed by patients who came for treatment or were referred by other dentists due to severe alterations. Moreover, in many cases it was not possible to obtain complete information for the genealogical analysis, and the molecular genetic analyses are only recently underway.

Conclusions
Considering the low worldwide prevalence of this pathology, i.e., 1 in 14,000 cases of all types of AI combined, 6  Additionally, it provides more tools for the adequate diagnosis of this pathology, which will allow for early intervention by the professional and adequate preventive and restorative actions in a multidisciplinary framework.