Transcribed by Amit Amin September 15

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, 2014

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[Diagnosis and Treatment of Oral Disease] [34] [Genetics and Its
Relationship to Dental Caries] by [Dr. Bretz]

[1] [title of slide]
[Dr. Bretz] Ok. Is this the entire class? No. What a honor to have all of you. Im
going to make this as painless as possible. When I say important, please write it
down. Lets talk about the genetics of dental caries by employing the twin study
model. Are there any twins in the class?
[2] [Rationale]
[Dr. Bretz] So, why do we use twin study models to understand any disease? Why
is that being employed in this type of study models. We do that because if you want
to do any genetic associations w/ any specific disease or trait. If you scan the human
genome and recruit thousands of patients w/o having any clue of what youre
looking for youre going to spend millions of dollars and youre going to end up
short. By using the twin study model, you can screen for the contributions of
genetics or environment on a specific disease and save millions of dollars. So if there
is any genetic association, you can quickly check that out and save effort and money.
Its a powerful tool to look at the genetic insight into genetic components for any
specific disease or trait. The basic premises is that if you have identical twins that
are 100% genetically similar and youre looking at the color of the hair and thats
highly correlated among the identical twins and not so correlated among the
fraternal twins who are 50% genetically similar than you have some indication that
there is a genetic contribution in this case as the color of the hair. You can do this
for any disease. For blood pressure, dental caries, any medical condition. When you
screen it, and see that a specific condition is highly correlated among identical twins
and not so correlated to fraternal twins, youre going to see a genetic contribution
to that specific disease or trait. If the correlation is not there among identical twins
then the environment has taken over and its effecting that specific disease or trait.
[3] [Important considerations]
[Dr. Bretz] Very important in our case, Im going to use dental caries as the
example to assess effect modifiers. If youre looking at dental caries, your population
has a lot of exposure to fluorides, antibiotics. They attend the dentist on a regular
basis. That is effecting the natural course of the disease. If you can have a population
as clean as possible w/o those effect modifiers that would be ideal to study dental
caries.
[4] [Age of Cohort]
[Dr. Bretz] What I just mentioned is very important by the way. The age of the
cohort. When would you conduct studies of dental caries. At what age? When the
teeth are erupting into the oral cavity. That would be the ideal time. If the surfaces
are fresh, any true genetic effects are going to be looked at w/ more precision at
those times. As the teeth age, then the environment is going to take over and its
going to be more difficult to look at any genetic associations.
[5] [Measurement of Phenotype]
[Dr. Bretz] Measuring the phenotype. The caries phenotype. If you conduct studies
looking at blood pressure and youre measuring it the wrong way, youre not going
to derive any meaningful conclusions. Same thing w/ dental caries. How many of
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you are familiar w/ quantitative light fluorescence techniques to measure dental
caries. Have you been exposed to this kind of information before? Quantitative light
fluorescence? This is a very extensive and powerful tool that can diagnose caries at a
subclinical level. You can monitor progression and remission of lesions over time in
a very precise way. More, or the better we can do in terms of measuring the
phenotype the better we will be in deriving a meaningful conclusion. A lot is missed
in terms of association in genetics and environment on a specific trait.
[6] [Statistical Analysis]
[Dr. Bretz] Statistics is very important. We have powerful tools now to look at
heritability estimates some of which I will show you in terms of results. The way
they are derived require pretty sophisticated methodology.
[7] [Study Site]
[Dr. Bretz] Years ago, I was looking for along w/ many researchers I was looking
for a population where I could conduct studies w/ the absence of effect modifiers.
We found a city in Northeast Brazil was where there was an abundance of twins.
Where water fluoride levels were less than optimal. Children were of a low
socioeconomic status. Virtually, when you first looked at them, about 91% of them
had never seen a dentist. It was a virtually an ideal population to look at genetics of
dental caries.
[8] [Study Population]
[Dr. Bretz] I mentioned this before. Weve collected over the years (10-12 years) a
variety of samples. Lot of information on many traits. Not only dental caries.
Microbiological/ immunological analysis. Occlusion topography. We conducted a lot
of studies and then b/c as we had access to them its so difficult to have access to
these populations you want to conduct or collect as much information as possible.
This is a very small example of the types of data we generated w/ these children. All
of them related to dental caries. We can look at the relative contributions of various
factors on caries itself.
[9] [Rationale]
[Dr. Bretz] One thing we learned the hard way as we were examining these twins
on a given day wed look at 80-90 families and theyd love to fool you when youre
dealing w/ twins. Scientist were doing there best and we were totally wrong b/c at
the end of the day twin a was twin b and when youre doing genetic analysis youre
data is all wrong. We came up w/ something that I would suggest for those who are
going to conduct studies in any area, not only w/ twins. We did something very neat.
[10] [Overview]
[Dr. Bretz] B/w the irises of twins are not identical. One of the few features that
are not identical in monozygotic twins. We scanned their irises. Every time they had
an exam, we would scan their irises and make sure that Individual A was Individual
A and B was B. But we learned that the hard way. So there is abundance of methods
as you well know that you can scan the iris and get precise information in terms of
identity.
[11] [Zygosity]
[Dr. Bretz] Another important information is that many of the studies in twins as it
relates to dental caries in the past were done by visual examination. The
investigator would say those are identical twins, those are fraternal twins. 5% of the
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time they were wrong and we employ assessment of zygosity. So conducting or
scanning highly polymorphic DNA loci is important to determine zygosity.
Otherwise you can be wrong at least 5% of the time and youre going to mess up
your data as well.
[12] [Dental caries]
[Dr. Bretz] Let me give you some examples of some studies. I dont want you to
know the statistics, I just want you to know how you employ the twin study model
and how you derive heritability estimates and if they are low, moderate, or high and
how we dissect genetics in the environment. This study we looked at dental caries
and microbial acid production in twins. We looked at 400 pairs of twins at the age of
1.5-8 years old. We preformed caries analysis and we also looked at the severity of
the lesion, not only if they had the lesions but also how deep they were. How severe
the lesions were.
[13] [Variables]
[Dr. Bretz] Came up w a number for each one of the twins were Twin 1 had x
number of decay surfaces divided by x number of total surfaces present. Came up
w/ the lesion severity index where we balanced the depth of the lesions throughout
the dentition. Each individual had a number so you could correlate among the
identical and fraternal twins how correlated those lesions and prevalence rates
were to see if they were heritable or not.
[14] [Methods]
[Dr. Bretz] We measured microbial acid production with a test that measured
lactic acid and you could score from 1 to 9 w/ 1 being low production and 9 being
high production. Each twin had a number that you could calculate correlations.
[15] [Results]
[Dr. Bretz] In doing so, you can see that the heritabilities, those are the ultimate
measures or estimates for caries prevalence rates were about 76%, lesion severity
index were 70%, and for the microbial acid production about 16%. 76 & 70% were
high suggesting there was a high genetic component. As expected, microbial acid
production was environmental. Acid being produced by bacteria in the mouth. This
is the (some word I cant understand) of the twin study model. If you look at caries,
you can then start being more detailed about looking at associations, dentitions,
surfaces, arches, and segments of the mouth and start looking in more detail. You
can then go back to the genome and scan for areas of interest. Thats a screening tool
and we used 400 pairs of twins. Had we employed families we would have needed
thousands to come to the same conclusions.
[16] [Longitudinal Assay]
[Dr. Bretz] Then we did just as an example, I mentioned about age being an
important consideration for studies of dental caries.
[17] [Methods]
[Dr. Bretz] We looked at heritability estimates of changes in dental caries
occurrence over time. We measured caries at baseline and we looked at the same
twins a year late. We looked at progression and remission of caries lesions over
time.
[18] [Results/Conclusions]
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[Dr. Bretz] Although not huge, yet significant. At the age of 1.5-3 the time that the
teeth are erupting into the oral cavity, you see the heritability estimates for caries
rates, lesions severity index, caries for occlusion surfaces, and deep dentinal lesions
they are some how moderate to high and very significant. So the teeth came into the
oral cavity at the age of 4-6 years old those heritability decreases sharply. What
happens here is that the environment is taking over and you dont see genetic
associations. At the age of 6 years those heritability estimates then go back to
moderate or high. What is happening now? 1
st
molars and incisors are coming into
the oral cavity. New teeth are coming in and the heratibilities become harder.
Proving the point I had made before about the age being important in looking at
genetic effects of dental caries.
[19] [Enamel Defects/ Objectives]
[Dr. Bretz] Going to share w/ this something about what I just mentioned is very
important. I wanted to mention about the heritability of enamel defects and
something here that I will show you is going to surprise you.
[20] [Methods]
[Dr. Bretz] We looked at enamel hyperplasia. We looked at enamel opacities and
mild fluorosis. If you were in a population that had less than optimal levels of
fluoride in the water supplies how do you have fluorosis in that population? Why
would that be? They eat toothpaste. They ingest toothpaste. Source of energy, of
food. Eating toothpaste. It took us a while to find that out but indeed as we
documented this later on, there was high ingestion of toothpaste.
[21] [Result]
[Dr. Bretz] Hence prevalence of hyperplasia about 4% in a cohort of 400 pairs
and the enamel opacities were 7.6% and the mild fluorosis 8%. You look at the
heritability estimates for all of these enamel defects were extremely high especially
for mild fluorosis. What did you learn about fluorosis or have you learned about it
yet? When we ingest high levels of fluoride at the time of tooth formation youre
more likely to develop fluorosis. We challenged this and were looking here and
showing there is a genetic component to fluorosis. Indeed very elegant studies
conducted in animals shows that if you gave the dosage of fluoride to a strain of
mice and you give the same dosage of fluoride to another strain of mice, one strain
develops fluorosis while the other does not proving that it has a genetic component.
Since those studies came out, many clinical studies in humans have proven that as
well. You can see that this is proof as well that fluorosis has a genetic component.
People can be more or less susceptible when exposed to the same dosages of
fluoride. This is important. This is something new we are learning here.
[22] [Sucrose Sweetness]
[Dr. Bretz] We conducted heritability estimates of dental caries and sucrose
preference. Here we are looking at if caries have a genetic component as we did
before and now we can submit those subjects to taste preference exercise for
sucrose and give them a number as well.
[23] [Methods]
[Dr. Bretz] Ill show you really quickly. We looked at caries rate like I showed you
before and then we submitted them to 5 concentrations of sucrose in grape group
and they were asked if they liked it, neutral, or disliked it the concentrations that
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they were submitted for. Based on that you can attach a number to each twin and
see how correlated the taste preference for sweetness was for each one of them.
[24] [Results]
[Dr. Bretz] We looked then again, the heritability estimates confirming what we
had seen before for caries prevalence rate 64, 61% and for sucrose sweetness
preference about 55%. Not very high but moderately high. Indeed since then, people
have looked or reported some genes involved w/ sweetness preference. What we
have seen clinically w/ the twin others have reported by scanning the genome
confirming what we had seen previously.
[25] [Microbial Colonization]
[Dr. Bretz] Then lastly, I wanted to show you something that I think that is highly
interesting. We looked at those twins and sampled their microbial flora from dental
plaque. You see that on the right side of the screen there is a pattern of bacteria that
is distinguishes health from disease. There are microbial components that will
dictate health and disease. What fascinates me here is that this cohort is from a low
socioeconomic status yet 25% were caries free. That interests me more than the
disease itself. Its to understand health giving the situation that these people were
in. This was across strata so from various age groups. Various age groups. We were
interested in looking at health from a microbiological perspective. These people are
eating poorly yet they dont have decay. How come?
[26] [Methods]
[Dr. Bretz] We collected plaque samples from diseased children, caries free
subjects, and we see that there is a patter where you see red.
[27] [No title]
[Dr. Bretz] Those are all beneficial species. Species that are associated w/ health.
Those in green are all species that are associated w/ caries, the disease. Since we
had this fraction of subjects that were caries free we were interested in looking at
heritabilities, how comparable these levels of beneficial bacteria were in identical
and fraternal twins.
[28] [Results]
[Dr. Bretz] You see that for all the beneficial species you can see that they are
moderate to high and very significant. They are showing that modulation of the
beneficial flora that are comparable w/ health had a heritable component. Then we
went one step ahead. We looked at these clinically and now were going to the
genome.
[29] [HLA]
[Dr. Bretz] We looked at SNIPs for HLA genes. Genes that modulate bacterial
colonization. Sure thing, many of the SNIPs of HLA genes were associated w/ those
beneficial species. This is the sequence of events. You screen clinically, and then you
go to the genome and you confirm or refute what you are seeing clinically. You
looked at how heritable the beneficial species were, you looked at the HLA genes
associated w/ modulation of n colonization of bacteria in the mouth, sure thing the
SNIPs for those genes were associated w/ the beneficial species. Any questions so
far? This was important by the way. This is just a second table showing the same
associations w/ another group of beneficial bacteria. Bacteria that are beneficial w/
health. Any questions so far?
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[30] [Ongoing work]
[Dr. Bretz] Weve been looking, I can spend many days here w/ you looking at
various sources of information. We will look at occlusion topography, enamel
defects, erosion, a variety of traits to see how heritable those traits are and how we
can make association w/ dental caries. I showed you sweetness preference,
microbial polymerization, enamel defects, dental caries, and microbial acid
production. You can look at many variables/ traits related to dental caries and come
up w/ an equation that we will have that will guide us in terms of establishing risk
for a given patient. We perfect the phenotype of measuring caries over time. Thats
highly important. The better we measure it, the more precise we are. Im sure that
eventually we will have all of this methodology to conduct dental caries
examinations available to you as they become less and less expensive. Its going to
be highly important. We conduct gene expression studies not only w/ individuals
but also w/ the bacteria involved w/ dental caries to know now only who they are
but how they function in health and disease. Look at areas, genes, loci that would be
responsible for those functional capabilities. Lots of possibilities. Very rich.
[31] [Admixture Mapping]
[Dr. Bretz] One last issue before we finish here, this story that Im telling you plays
very well w/ the Brazilian population. It may not be true in Norway. In Norway, they
are Vikings that are conserved genetically. In Brazil, a mixture of races is so big that
the stories can be totally different. So important to replicate the studies that I just
mentioned to you in other populations to see if it plays out like it played in a
population that has a very rich racial background unlike the Norwegians. Compare
studies, results to see if the story plays just as well otherwise what I was telling you
would be relevant in a Brazilian population but not so in a Norwegian population
but its highly conserved genetically. Ok. We are conducting these comparison
studies as we speak.
[32] [Collaborators]
[Dr. Bretz] This is years and years of collaboration. A lot of people involved. Im
showing to you a fraction of the studies we have conducted in hope that this has
been information. Thank you, any questions? Doubts?
[33] [title of slide]
[Dr. Bretz]