Genomics: Insight

Research Question: What genes affect enamel formation and inherited phenotype?

Amelogenesis Imperfecta 

Tooth rot, infected tissue and bone, lumpy teeth, tooth resorption, dental caries, and gum disease are all possible symptoms of amelogenesis imperfecta (AI). These set of rare diseases changes the structure and strength of tooth enamel leaving the teeth discolored, deformed, and weakened by malformed enamel. It is a heterogeneous group of genetic disorders caused by proteins not being able to complete their functions during the enamel formation process called amelogenesis¹. There are two types of AI which will be discussed that refer to the two major enamel issues. The most common type of AI is Type 1 and is characterized by hypoplastic enamel which is a reduction of enamel leading to small crowns, grooves in the teeth, and an open bite. Type 2 is characterized by hypomatured enamel where the enamel is too soft or too brittle leading to a pitted texture as well as breakage and sensitivity. AI is estimated to affect 1 out of every 14,000 people in the US, but in other populations the frequency is varied². This confirms that there is a hereditary link, but due to the large range of issues and symptoms, it can be difficult to identify what genes lead to which issues. AI creates pain, discomfort, and even social isolation, so discovering genetic causes can help affected individuals determine the probability of offsprings also having symptoms leading to specific enamel restoration care and other treatment options at an earlier age.

Through genomic research, scientists have found multiple proteins that work during enamel formation. These are encoded by different genes that when mutated can cause AI because the proteins are not able to fully complete their functions. There are three main genes connected to AI that have been researched thoroughly.

AMELX

One gene identified to cause AI is the amelogenin gene, AMELX which is located on the X chromosome³. Amelogenin is the most abundant enamel matrix protein and when mutations appear, the proteins become misshapen which means they can not carry out their function leading to the reduced thickness (Type 1 AI) and reduced hardness (Type 2 AI)⁴ of the enamel. In one study, exon analysis, splicing assays, immunoblotting, and RT-PCR on six families with AI determined which mutations lead to which phenotypic expression⁴. The research leads to the idea that the deletion or frameshift changes the protein to such a degree that they cannot function, marking these as amorphic mutations that cause hypoplastic and hypomaturation enamel. The presence of enamel, however, points towards amelogenin working on building the outer surface of enamel4 up rather than actually starting formation of enamel. Another study focused on a specific transition mutation and its alternative splicing. This led to the result that although the mutation is what causes AI, the different splicing variations can impact the phenotype for the whole family³. Amelogenin is an important protein for enamel formation, and so being able to link AMELX mutations to phenotypes seen in families allows for advancement in dental understanding because there is a firmer baseline of information about the disease and what causes it.

ENAM

ENAM is another important gene, encoding for the protein enamelin, that builds tooth enamel by continuously developing crystallites on the outer surface of teeth to provide strength and durability⁵. There are at least 14 mutations identified to cause AI from this gene, which can have either autosomal dominant or autosomal recessive heritance patterns. Often, with autosomal dominant traits because there is only 1 copy of the mutated gene, the effects are less intense because some of the correct protein is being made, but patients will still experience hypoplastic enamel such as small grooves or pits in the tooth enamel. With autosomal recessive mutation, there are 2 copies of the mutated gene resulting in no production of the proper protein and more severe AI.This makes the protective covering extremely thin or absent making the teeth weaker and more prone to other oral diseases. This idea that phenotypic severity is related to the amount of functional proteins has been confirmed by a study looking at mutations of ENAM and their corresponding phenotypes1. A correlation was observed between biallelic mutations and severe hypoplastic as well as heterozygous mutations having fewer affected areas on the tooth surfaces1. All this information helps piece together how mutations of genes affect the actual processes happening in the body as well as informing about different areas to look into for future research.

AMBN

Ameloblastin is another extracellular matrix protein that works in cell signaling and developing enamel⁶ and is coded by the gene AMBN. This protein is very important for enamel formation because it begins the process of amelogenesis by secreting the first layers of enamel. If a mutation disables the function of ameloblastin, the enamel will not have a good starting layer for proteins like amelogenin to attach to, causing AI. For years the consensus was that any mutation to the AMBN gene was detrimental, but a recent study determined only two mutations of AMBN actually cause AI⁷: a homozygous genomic deletion and a homozygous splice junction defect, both causing autosomal recessive Type 1 AI⁷. Another important aspect of this study is that researchers were able to find deeper understandings of these genes’ function leading them to discover that AMBN mutations are not a major cause of AI. This allows dentists and researchers to recognize that AI is primarily caused by malformations of the outermost layer of enamel and more focus should be spent exploring direct connections between ENAM or AMELX and phenotype.

Conclusion and Next Steps

AI is controlled by many other genes that can affect the severity of the disease, but the AMELX, ENAM, and AMBN genes have been proven to have direct phenotypic results. Genomic studies continue to both identify new mutations that cause AI, and gain a better understanding of previously identified mutations that do not cause AI, leading to a fuller picture of the disease and ways to combat malformed enamel. When AMELX is mutated the outer layer of enamel is thin and weak. When ENAM is mutated the enamel is very weak and severity depends on if there are any functional proteins to partially do the work. When AMBN is mutated the enamel cannot form correctly, but it is now known that for this gene only a few specific mutations actually lead to AI and thin enamel. There are so many places for enamel formation to go wrong, but over time there is more information about which mutations and alternative splicing patterns lead to which specific phenotypes. For future research on AI, the protein tuftelin would be a great place to look into because it helps in enamel mineralization. Currently researchers think there could be a connection between AI and tuftelin, but there is not much literature about its sequence or mutations. Looking forward, more people affected with this disease will be able to do genetic testing to identify which mutated genes they carry to determine the likelihood of future offspring having AI. This can give those families and their dentists the insight to start specific enamel restoration care at a very young age. Learning more about AI through the different possible causes can lead to better treatments and less pain for future generations.

"AMELX, ENAM, and AMBN genes have been proven to have direct phenotypic results."

References 

1. Wang Y-L, Lin H-C, Liang T, et al. (2024), ENAM Mutations Can Cause Hypomaturation Amelogenesis Imperfecta. Journal of Dental Research. 2024;103(6):662-671. https://doi.org/10.1177/00220345241236695     
2. Smith, C. E., Poulter, J. A., Antanaviciute, A., Kirkham, J., Brookes, S. J., Inglehearn, C. F., & Mighell, A. J. (2017). Amelogenesis imperfecta; genes, proteins, and pathways. Frontiers in physiology, 8, 435. https://doi.org/10.3389/fphys.2017.00435
3. Young-Jae Kim, Youn Jung Kim, Jenny Kang, Teo Jeon Shin, Hong-Keun Hyun, Sang-Hoon Lee, Zang Hee Lee, Jung-Wook Kim, (2017) A novel AMELX mutation causes hypoplastic amelogenesis imperfecta. Archives of Oral Biology, 76:61-65.      https://doi.org/10.1016/j.archoralbio.2017.01.004.  
4. Wang, S. K., Zhang, H., Lin, H. C., Wang, Y. L., Lin, S. C., Seymen, F., Koruyucu, M., Simmer, J. P., & Hu, J. C. (2024). AMELX Mutations and Genotype-Phenotype Correlation in X-Linked Amelogenesis Imperfecta. International journal of molecular sciences, 25(11), 6132. https://doi.org/10.3390/ijms25116132
5. Daubert, D. M., Kelley, J. L., Udod, Y. G., Habor, C., Kleist, C. G., Furman, I. K., ... & Roberts, F. A. (2016). Human enamel thickness and ENAM polymorphism. International Journal of Oral Science, 8(2), 93-97. https://www.nature.com/articles/ijos20161
6. Natalie C. Kegulian, Gayathri Visakan, Rucha Arun Bapat, Janet Moradian-Oldak, (2024) Ameloblastin and its multifunctionality in amelogenesis: A review,Matrix Biology, 131: 62-76. https://doi.org/10.1016/j.matbio.2024.05.007
7. Liang, T., Hu, Y., Smith, C. E., Richardson, A. S., Zhang, H., Yang, J., Lin, B., Wang, S. K., Kim, J. W., Chun, Y. H., Simmer, J. P., & Hu, J. C. (2019). AMBN mutations causing hypoplastic amelogenesis imperfecta and Ambn knockout-NLS-lacZ knockin mice exhibiting failed amelogenesis and Ambn tissue-specificity. Molecular genetics & genomic medicine, 7(9), e929. https://doi.org/10.1002/mgg3.929