Discussion of Session 9

Amelogenesis imperfecta Moderator: Tim Wright

Wright: Jan (Hu), would you please comment on yourthoughts regarding the impact or benefit of finding adisease-causative gene for the patient population.Hu: I get this question very often from people whether

they are clinical faculty or residents or students, �Why areyou doing this? Isn�t it just a mutation?� I think, �It�s notjust my personal passion and interest. For this family it�svery important for them to find out what�s really wrongwith them.� So, identifying the cause, as I�m trying to do,brings comfort to them. It�s extremely interesting to usthat using either mutational analysis or the newest ge-netic approach is providing a great opportunity toidentify new genes that are responsible for enamel for-mation. Most importantly, from these various types ofmutations, we can deduce how the mutations might af-fect protein functions, and we�ll be able to see thesestructural defects. So, we also collect these teeth from thesubjects and try to analyze their enamel structures as wellas their dentin structures. And I think, most importantly,if one day we are able to have genetic therapy for theseindividuals, it�s important that we are able to make thecorrect diagnosis, and to know the defect of a specificgene so that effective replacement can be delivered tothese subjects.Wright: So, you�re showing basically a deletion, com-

plete deletion, of amelogenin, X-linked (AMELX), right,in one of your families?Hu: Yes. That�s right.Wright: There are other reported AMELX mutations

similar to the one you are reporting, some of which havesignal peptide mutations, and we assume that they don�tproduce any protein and the first reported AMELXmutation was basically an AMELX deletion. The phe-notype on that AMELX deletion was a little back andforth. With the one you describe, and I don�t know if youstudied a man or a woman, but I believe you are callingthe phenotype hypomaturation-hypoplastic. So, myquestion, and I don�t know if you�ve had a chance tolook at the teeth, but I�ve always wondered, if you knockout amelogenin in a mouse, you really don�t have muchenamel. So, if you have something similar to an AMELXknockout in the human male, how do you get such adifferent phenotype? Because it looks like your pheno-type is a little less severe than some of these reportedsignal-peptide mutations or AMELX deletions. Is theenamel very thin as in those previous reports, or is it notas severe? Do you think we have a contribution fromamelogenin Y-linked (AMELY), potentially making thephenotype less severe?Hu: The Y chromosome copy of amelogenin (AMEL)

was present because it gave us a lot of trouble initially

when we tried to characterize the mutation. In terms ofclinical phenotype, we really didn�t see anything moresevere than reported for other families in the literature.The number of AMELX deletions is very limited. How-ever, Carolyn Gibson has a family with a large segmentof gene deletion in the X chromosome, includingAMELX and many other genes, such as the ARHGAP(RhoGAP) gene. This family has all kinds of systemicproblems. In our family, at least by report and evalua-tion of cranial facial structures, we don�t see anydetectable anomalies. The tooth phenotypes seem to bethe only things that are wrong, and the enamel is verythin, but you can see a hint of the enamel being a little bitwider in the cuspal area and the marginal ridge.Paine: Michael Paine, University of Southern Cali-

fornia. Jan, with these new mutations that are coming upand the associated problems, such as gingival hyperpla-sia, is it still reasonable to use the term amelogenesisimperfecta (AI) because that term was originally used todescribe enamel defects without any associated defects?Hu: I would like Tim Wright to come back on because

he is the expert on that and we respect his opinion. Butone thing I would say is that for most of the families thatwe characterize, we have to ask, very carefully, as muchas possible and observe as much as possible to hopefullydraw out any minor systemic or other organ or tissuedefects. In other words, in the families that we charac-terize, we want to be able to feel confident that this isactually an enamel defect. Would you like to commenton that Tim (Wright)?Wright: It�s a sticky wicket. You know, the more you

look at, and carefully evaluate, the phenotypes the morelikely you are to find things. Even in the �classic� AI casesthat are enamel only, it�s not uncommon to find eruptiondefects, or other effects. So, we ask, �are these featurespart of the primary defect or are these secondary effects?�Skeletal open-bite malocclusions are highly prevalent inAI, are associated with different genes, and are probablynot a primary gene effect. I do believe that, in a recentpaper on AI, they showed gingival expression of thecausative gene. So, is gingival hyperplasia a primary ef-fect or a secondary effect? A lot of AIs have gingivalanomalies. I think that the more we understand aboutthese conditions, the more it will become evident thatthere are gray areas and overlaps. So, you�ve got con-ditions like Jalili syndrome with the eye associations, andthere are numerous reports in the literature where theyreport a systemic condition and AI. I tend to be a bit of apurist and try to keep AI to describe conditions that areprimarily only enamel. If you call everything with enameldefects amelogenesis imperfecta, the term becomes rela-

Eur J Oral Sci 2011; 119 (Suppl. 1): 338–341DOI: 10.1111/j.1600-0722.2011.00933.xPrinted in Singapore. All rights reserved

� 2011 Eur J Oral Sci

European Journal ofOral Sciences

tively meaningless. It�s like calling every bone defectosteogenesis imperfecta. Ultimately, I think we reallyneed to have a new nomenclature that incorporates themolecular features and includes the cardinal phenotypicfeatures. At this point in time, even with only 50% of theAI genes known, I think we could probably do a muchbetter job with the classification that would communicatemore effectively than what we currently have. We�re duefor an international consensus on coming up with a newclassification that includes the molecular basis.Simmer: Jim Simmer, University of Michigan. I want

to make a comment on that also when you�re screeningpatients, you often don�t notice the non-enamel defects.They can be subtle or they can even be non-existent. But,in most cases, they aren�t. For instance, collagen typeXVII (COL17) is a gene considered responsible forjunctional epidermolysis bullosa, a recessive condition.However, there is a case where only one allele was af-fected and they had straight AI with no other manifes-tions. So, this is a case that was actually one of the first 24AI families we evaluated, and so it was originally classi-fied as AI in the scheme, and then you find a mutationand then that gingival hyperplasia looks a little moredramatic. We were aware of it but when the patient comesin you don�t know if they have nephrocalcenosis orsomething else that�s not obvious. So, often you have toexpand the definition because you become more aware ofadditional symptoms. So, it�s never going to be com-pletely narrowly defined because you just keep coming upwith families that you diagnose as AI and who actuallyhave multiple conditions that you don�t notice at first.Hu: Well, I would like to add to that, with this

particular FAM20A mutation other than in gingivalhyperplasia, there are also dental impactions as well asgallstones associated with that mutation. At this time, wedon�t know whether these are primary effects of the genemutation or secondary effects. I think that it is good tojust keep an open mind. It is the same thing, along thesame train, the open-bite association with an AI that onaverage is a little bit more accelerated than tooth erup-tion associated with AI. These are being observed andrecorded more frequently in the literature. So, I want tokeep our minds open.Le: Thuan Le, San Francisco. Tim mentioned about

nomenclature and I have a question that, often inposterupted teeth, the terms hypocalcified, hypominer-alized, and hypomaturation are really confusing. It isnearly impossible to distinguish the difference. Some-times you can distinguish hypoplastic, but it is very dif-ficult to distinguish the other two I mentioned. As anexpert, how can we have a better definition of what ishypocalcified or hypomineralized vs. hypomaturation?Hu: Hypoplastic means enamel is thin. So, that one is

relatively easy. Clinically or radiographically you shouldbe able to discern that the thickness is not normal. Thehypomaturation one is a little harder. It should be fullthickness, by definition; however, its degree of mineral-ization is decreased and therefore radiographically, theenamel layer will not contrast well with dentin. Thehardest one that I always have a problem with is hypo-calcified.

Wright: These classification categories were developedby Carl Witkop, Jr., and they were based on the mech-anism as he perceived it back in the day. He started hisclassification back in the 1950s and early 1960s and itturned out to be pretty insightful. If you look at what hecalled hypomaturation, we now know that there arematurational defects, either with amelogenin that is notapparently properly processed based on interruption ofcleavage sites and the proteinase changes. The hypocal-cified group, I always had trouble getting my headaround too. Witkop said that this was a problem withthe initial mineral formation and nucleation that nevergot off the ground properly and then the enamel nevermineralized properly. As we don�t know the mechanismfor FAM83H, we don�t know if he was correct. I thoughtit was pretty impressive that he was correct on the otherAI types because this was pre-molecular biology. Therereally wasn�t a lot of protein chemistry at the time. Thiseven pre-dated the term amelogenin. What I tell clini-cians is, if you can discern whether it�s thin or it�s hyp-omineralized, from a clinical point of view, that�s criticalin decision-making. For proper diagnosis and classifica-tion you want to be looking at the inheritance pattern.When searching for genes, you are looking at familyhistory and phenotype. That�s what is going to direct youto the most likely genotype. From a classification pointof view it might just be better just to call them all hyp-omineralized and, in fact, in a lot of European literaturethey�ve done that.Le: Looking at teeth posteruptively, where there is

attrition and staining it can be really difficult to deter-mine the phenotype.Wright: That is why, when possible, you look radio-

graphically at the crown shape of unerupted teeth to seeif it looks normal. If you don�t see enamel then it ishypoplastic. If the crown form looks normal but there isno enamel contrasting to dentin then it is hypomineral-ized. Then look at inheritance, because the hypocalcifiedtypes are most often autosomal dominant and currentlyall of the proteinases associated with hypomaturationAIs are autosomal recessive.Zhang: Jan Zhang, Johnson & Johnson. After you

identify these families with their problem, what do youdo to help them?Hu: It is a very long process that starts in the primary

dentition. The most difficult time is probably the tran-sition stage when patients are 6–12 years of age and theprimary teeth are shedding and adult teeth are coming in.Personal appearance becomes very important and res-torations are therefore critical. Fortunately, there aresome financial aid resources available to help these pa-tients at this age and most can be treated using availablerestorative material like composite and metal. When theyget into the permanent dentition a full reconstruction isplanned. Many patients get so frustrated that they justgive up and elect to have full-mouth extraction anddentures. For those fortunate enough to have thefinancial means to go through full reconstruction, thecost is about $60,000.Hubbard: Mike Hubbard, Melbourne. I�d just like to

reinforce what Tim (Wright) said. I think that is there a

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basis in other literature now, that if you don�t know whatit is, you should call it hypomineralizationation? I thinkthat�s the umbrella term, and then under that fall twocategories: hypomature (where we have residual amelo-genin or enamel protein) and hypocalcification (wherethe amelogenins have disappeared). These days it is veryeasy to determine this by scraping an enamel lesion andrunning a gel. So, I think we should be heading towardsa molecular diagnosis at the protein level.Beniash: Elia Beniash, University of Pittsburgh. Jung-

Wook (Kim), if there is a really serious phenotype ofenamel, then the dentin might be exposed to osteoclastswhich normally resorb the roots. The osteoclasts mightthen consider the crown of the erupting tooth as thetarget and they might simply just chew it up, providing amechanism for crown resorption in AI. What do youthink?Kim: Yeah, it�s possible.Bartlett: John Bartlett, Forsyth. Jan (Hu), can you

speculate on why the absence of the expression of aprotein would cause apoptosis?Hu: I don�t think I could begin to speculate at this

time. However, I�m happy that this presentation is fol-lowing Jennifer�s (Kirkham) presentation, because basedon her study, it does give us some hints that perhaps thematrix secretion is not normal and the accumulationoutside of the cells, in between the cells, could be a resultof ameloblast cell layers being altered. That could be aresult of changing the cell matrix interactions. It could bea result of altered cell adhesion and attachment, andbased on Jennifer�s study, we will go back and check outthe transition stage to see whether there�s a hint ofaccumulation within the cytoplasm that could be con-tributing to the cell morphology.Simmer: This not a question; it is more of a comment.

From each of these presentations, we could see thatwhere there is a mutation affecting protein production,or a lack of production, there often seems to be a sig-nificant cellular response. If the situation becomes so badthat perhaps there is an immune reaction leading toresorption, endoplasmic reticulum stress response, orjust simply to crowding of the cells, leading to compli-cations that would lead to cell death. Now, when we arethinking about mutant enamel proteins, we are moreaware of the cellular aspects of these mutations. Do weneed to change our experimental approaches and per-haps consider more the in vivo situation and the effect onthe cell, irrespective of any changes in the mechanism ofenamel formation, considering the pathology in cases ofAI.Kirkam: Jennifer Kirkham, Leeds. Yes, I think there is

still room to be thinking about in vitro work to help us toput all the jigsaw pieces together. I think the lesson that ittaught me is that this puts the way we are thinking, interms of not dividing off the extracellular compartmentin our thinking from the cellular compartment, but to bethinking of it in a holistic way, because that�s the waytissue works. We shouldn�t be trying to divide it up,really. And it maybe just brings it into line with every-thing else. When you look into other inherited connectivetissue disorders, mutations in extracellular matrix pro-

teins there can exert their major effect intracellularly, so Iguess we shouldn�t be surprised that it is the same, insome cases, for enamel. My own feeling is that we shouldbe thinking about this in a holistic way, or thinkingabout the cells and the matrix as part of one whole,rather than dividing it into two separate compartments.Hu: There is value to in vitro studies and we can def-

initely learn from different studies to provide a betterdesign for our in vivo studies, which are usually moretime consuming and more involved. Therefore, I believethat it is important to have both approaches.Robinson: Colin Robinson, Leeds. I would like to ask

about interpretation of the phenotypes in terms of thepossible secondary effects because you all really inter-preted phenotypes in terms of what has happened to theproteins that have been mutated. When you are eruptingporous enamel with a lot of different proteins in it, isthere the possibility of uptake of albumin or salivaryproteins that will stop crystal growth and once it is in themouth might the saliva itself and the plaque biofilmchange posteruptive mineral uptake? What is your feel-ing about that in terms of, do you see some of that, or isit purely caused by the mutated protein?Hu: In terms of humans we do not have the privilege of

obtaining their teeth for protein analysis. For most ofour mouse group, however, we have to be very carefulwhen assessing the matrix component. We usually ana-lyze those teeth pre-eruption, and we have to dissect outthe tissue very carefully to minimize contamination fromthe surrounding structures. But, you are right. From ourexperience of analyzing pig teeth there is usually signifi-cant blood protein contamination, so we are aware ofthese issues and therefore, when we analyze our knock-out mouse model, we have to be very careful to select theproper stage for protein work.Robinson: In some patients, for example, we�ve seen

from Tim�s (Wright) work, how much of a secondaryeffect was caused by, for example, mineral accumulationfrom the saliva?Hu: From humans we always see, especially the adult

hypocalcified type, significant plaque and calculus on thetooth surface, and therefore I can imagine that if youwant to study the protein component of these teeth, youhave to be very careful when treating those teeth prior toactually extracting the matrix component.Le: I have a question for Jan (Hu) also. We now know

that your group and Tim�s (Wright) group have a veryinteresting mouse knockout model and LacZ knockinmodel. So, my question is, when you do the replacement,do you keep the sequence that codes for the signal pep-tide on enamelin?Hu: I think this replacement started fromATG. Prior to

that is the normal sequence plus the promoter sequence.Le: But then are you able to detect the beta-gal

staining in the extracellular matrix?Hu: No. This is a nuclear-localized beta gal.Ton Bronckers: Jan, I have a question for you. That is

beautiful Von Kossa staining in the enamelin-nullmouse. I was really surprised to see how regular theameloblastic layer was in the null animal. And I won-dered, of course, it�s not that Von Kossa is not that

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sensitive. I was wondering, because there are still verysmall crystals present in that matrix and you didn�t pickit up. What we see in vitro, if you culture the tooth buds,is that very often you get to really have a mineralizedmatrix that is very amorphous and the Tomes� processesare very irregular, and at the dentin–enamel junctions,you see small prongs of crystals growing out from thedentin. So, in your case, I would have expected to seesome mineralization near the dentin–enamel junction;however, I didn�t. Could you comment on that, please?Hu: I think your speculation might be correct. We

have just started to produce transmission electronmicroscopy sections at earlier stages, when mineraliza-tion just takes place, and so we don�t have enough datato give you a good answer at this time. However, fromtime to time we could see some hint of crystal in pre-liminary sections of images of the null mouse. But theseare primarily where Tomes� processes would be, not onthe dentin side.Bronckers: Is there also a little bit of concern that you

really need enamelin, or can crystals initiate at the den-tin–enamel junction and then grow as long as the con-ditions are okay with some amelogenin to start? This isnot only the knockout with enamelin, but needs a veryspecial protein to get mineralization.Hu: It certainly looks like that. We don�t have enough

evidence to state that the presence of enamelin allowsinitiation of the crystallite at this time. However, it is truethat in our enamelin we don�t see crystal initiation in thenormal fashion.

Clarkson: Brian Clarkson, University of Michigan.Jan, we�ve been able to grow crystals in an in vitro situ-ation on the enamel–dentin junction without any prob-lem. I wonder whether anyone thinks that the growth ofthese crystallites is dependent on the morphology of theenamel–dentin junction. Have you, or has anyone else,looked at any difference in the morphology of the en-amel–dentin junction in any of the knockouts. Wouldyou just concentrate on what protein is there and whatisn�t?Hu: That�s a very good question. We have just started

to look at the dentin–enamel junction using transmissionelectron microscopy and we don�t have enough data tospeculate at this time.Wright: A question to Jun-Wook. Kim, do you want

to speculate on the significant phenotype difference ofmales vs. females with the same mutation – one withsignificant resorption and the other with none. What doyou think is going on with that?Kim: I think that the phenotype difference, even in the

same family, is a result of different dietary habits, dif-ferent diets, and different eating patterns, as well as thegenetic background, compound genetic diversity.Simmer:Not to let you off from that question so easily,

Jun-Wook! I don�t remember in your case – were both ofthe two affected males, or was one male and one female?Kim: Both were male.

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