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Ann Periodontol

Reections on the 20th International Conference on


Periodontal Research: Molecular Determinants of Risk
With a Commentary on Post-Genomic Periodontal Research
Musings of an Experimentalist
Steven Offenbacher
Center for Oral and Systemic Diseases, University of North Carolina, Chapel Hill.
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CONFERENCE OVERVIEW
The 20th ICPR was one of the first periodontal
research conferences of the post-genomic era. It was
organized with the purpose of providing periodontal
scientists with an overview of what the future may hold
using microbial and human genomics as tools to
explore pathogenesis and redene diagnosis, preven-
tion, and treatment. It brought experts in molecular
genomics and analytical modeling methods from dis-
parate areas to provide discussions ranging from inte-
grating genomic data into epidemiological risk mod-
els to designing laboratory experiments to explore
changes in transcriptome expression in cells, tissues,
and animal models. Elegant techniques were shown
that begin to fully characterize the biodiversity of the
oral biolm and to characterize the virulence genes of
organisms with genomic approaches. The goal of the
conference was to begin to bring these molecular meth-
ods of analyses to bear on risk assessment of peri-
odontal infection on oral and systemic disease. The
intent was to begin the process of identifying specic
genetic markers that confer risk or resistance, and cer-
tain microbial virulence genes that are critical to patho-
genesis to reconstruct denitions of diseases that are
not necessarily generalizable to the population at large,
but provide a detailed, specic characterization at a
patient level or within a smaller subset of individuals.
Papers were presented that provided new insights into
the thought processes that will be needed to establish
new clusters of clinical and laboratory ndings to dene
periodontal disease syndromes and to provide new
denitions of disease that better characterize the oral
condition as it serves as an exposure for systemic
complications. Commonalities of pathogenesis were
raised when periodontal disease was considered as a
triad of clinical signs, oral infection, and inammatory
response rather than a single cluster of clinical nd-
ings. These mechanisms extend from local tissue
destruction to pathways that involve the vasculature
and pregnancy. Papers on the inuence of lipid and
carbohydrate metabolism, as well as obesity and dia-
betes as metabolic conditions, were discussed as to
how they modify periodontal disease expression or
serve as systemic stressors. Discussions ranged from
lipid modulation of inammation to discussions of the
differences between anthropomorphic definitions of
central obesity, body mass index, and waist-to-hip ratio
on disease models. These papers and discussions
helped to clarify how obesity and diabetes serve as
effect modiers as one examines the role of oral infec-
tion on periodontal status and systemic complications
such as cardiovascular disease.
The research trend manifested by this ICPR meet-
ing is clear. We are rapidly moving into an era when
molecular laboratory findings, especially protein or
DNA-based, using host or pathogen biological samples
will be tightly integrated into our traditional clinical
denitions of disease. It is the promise of the future
that we will eventually be able to better dene the dis-
ease at a patient level to provide personalized med-
icine. That is, based upon our new complex diag-
nostic profiling that includes an individuals genetic
background, behaviors, and exposures, we will then
be able to offer a specic preventive or therapeutic
strategy that is customized to the individual. This Con-
ference was a successful step towards describing these
approaches to explore this formidable challenge of
personalized medicine. And, as a consequence of
reection and debate on this transitional thinking and
in the spirit of the ICPR, which traditionally fosters an
open-minded exchange of controversial ideas, the fol-
lowing personal commentary on this post-genomic
future is offered.
COMMENTARY
Many of us in science can remember the excitement
generated by the original scientific breakthrough of
Watson and Crick that started it all 50 years ago and
can now marvel at the realization of milestone achieve-
ments with the completion of the human genome pro-
ject and the recent sequencing of several genomes of
relevant oral pathogens. It is a phenomenal time to be
in science, as these shared national scientific land-
marks of achievement rival the collective effort of
putting a manned spacecraft on the moon. These new
genetic roadmaps should help us navigate our exper-
imental pathways as we try to decipher the complex
intricacies of microbial virulence and pathogenesis,
2009 3/26/03 1:12 PM Page i
Reections on the 20th International Conference on Periodontal Research Volume 7 Number 1 December 2002
gene-environment interactions, periodontal disease sus-
ceptibility and resistance traits, and the role of peri-
odontal disease as a systemic exposure. The magni-
tude of the breadth and complexity of the information
has birthed its own discipline (bioinformatics) and is
purported to eventually enable us to achieve the com-
mon goal of personalized medicine that includes den-
tistry. That is, the prevention and treatment of human
ailments will be repackaged not to deliver what gen-
erally works best and is globally safe for everyone, but
rather, in the context of optimizing the prevention and
intervention specically for the unique patient using
information about an individuals genetic characteris-
tics, exposures, biomarker patterns, and behaviors.
This is the promise of medicine for the future that is her-
alded by the omics. But exactly how the new post-
genomic era will create new directions for dental
research, the context in which dental scientists will con-
tribute to the future of molecular dentistry and the main-
stream of biomedical research, is just beginning to
unfold.
Inevitably, changing trends in research are precipi-
tated by new discovery and the creation of new tools.
Landmark technological advances that create new
methods can sometimes create self-validating scien-
tic lines of investigation rather than simply provide
better tools for scientic discovery. Thus, as we move
into this new area of genomics, one is reminded of the
afterglow of post-Apollo landing times when we glee-
fully discussed living on Mars, just as today with the
sequencing of the human genome some now forecast
the imminent end of human suffering from disease. At
times, this optimism is fueled by the ecumenical media
hype for one trendy research method or another. But
the history of biomedical research is spotted with exam-
ples of ineffective lines of investigation, suggesting that
sometimes the tools we use drive the scientic ques-
tions we pose rather than the other way around. For
example, with the perfection of electron microscopy,
structural biology became the science of the day, and
then there was the era of monoclonal antibodies. How
many research facilities still harbor antigen-specific
monoclonal cell lines that may have been of interest but
were never characterized? With the explosion in mol-
ecular biology over the last 2 decades, it seems that
virtually every scientific discipline has now been
renamed to include the word molecular. And in recent
years, how many laboratories have studied a unique
gene that has been cloned, sequenced, and, via trans-
genic models, been over- or underexpressed, and still
remains without a clear function?
With the sequencing of the human genome and a
growing list of microbial genomes providing the criti-
cal blueprints for potential experiments, some specic
thoughts came to mind. I would like to briey raise
some points of discussion and, perhaps, controversy.
These comments are not intended to criticize or dimin-
ish the tremendous advances that have been realized
by the milestone achievements of the past several
decades. Instead, these comments are made to pre-
sent a personal perspective that may, for some, pos-
sibly minimize well-intended experimental meander-
ings as we embark into the new post-genomics era.
1. My rst point is that the human genome sequence
was done on a composite individual to produce a lim-
ited sequence map for those genes that code for pro-
teins. Those open-reading frames are regions that are
transcribed into messenger RNA and then translated
into proteins. That represents only about 25% to 35%
of the entire DNA sequence. There are huge areas of
DNA in between these open-reading frames that are
thought to play some regulatory and structural role for
maintaining the genome. The data in the human
genome map contain, for example, the entire sequence
for type I collagen alpha chain. It is often popular to
cite in the literature that the differences between the
genes for man and a gorilla differ by less than 5%. It
is no surprise that the sequence of type I collagen for
man is almost identical to that of a gorilla. It has been
argued that the differences between gorillas and men
can be explained by that infinitesimally small, but
somehow magically transforming, 5% that represents
the tiny difference in the coding regions, as compar-
ing our 2 closely aligned species. From my perspec-
tive that concept is analogous to saying that tigers,
zebras, and convicts are almost identical that is,
from the viewpoint of black stripes. After all, collagen
is a basic building block of connective tissue and it
would be inefcient for us not to have utilized this pro-
tein as an evolutionarily conserved molecule. Clearly,
genes that code for proteins representing the common
building blocks of life that are needed for a biologi-
cally viable mammal should logically be quite similar.
These sequences are conserved because mother
nature found them to be useful so they need not be
reinvented. But what about those unknown sequences
contained within the non-coding DNA? Many years
ago these genes were termed nonsense genes, as the
gene products did not result in protein sequences and
there is tremendous diversity in these sequences. It is
the unique combination of sequences in this region of
the total DNA that is used to incriminate a suspected
murderer, for example. There are regions of DNA that
contain so-called random repeat areas of DNA that
do not code for protein but can be used to identify a
person uniquely and establish parentage and familial
linkages. However, it might be argued that it would
also seem totally evolutionarily inefcient that any DNA
sequence would be conserved, such as these non-cod-
ing regions, if they are indeed nonsense or non-func-
tional. It would seem more plausible that these
sequences are not, in fact, random and contain infor-
mation as yet to be discovered. These are the
sequences that we inherited from our parents that give
us our special personalities and physical features and
they are probably no more random than those regions
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Ann Periodontol Offenbacher
that encode for protein that we also considered to be
random gibberish just 50 years ago. One might recall
that prior to Watson and Crick, the entire 4-base
sequence of the entire chromosomal DNA was also
considered a random chain of bases. We now know
that it is clearly not random and that it has a critical
importance with regard to lineage and heritance. This
would suggest that there is much more of the genetic
code yet to be unraveled.
The limited nature of the DNA sequence decoding
data also raises questions regarding the general util-
ity of the human genome data to ascertain risk for dis-
ease and assess response to therapy. For example,
when forensic scientists seek to identify a person
uniquely, it is not done by comparing DNA from an
individual to the human genome sequence data. After
all, using just these data, we could not really distinguish
very efciently if the DNA sequence were from a mur-
der suspect or a gorilla. One might then easily ques-
tion how we are to use these sequence data to iden-
tify genes which confer risk for diseases especially
most of them that have familial tendencies like peri-
odontal disease, cardiovascular disease, and diabetes.
And what about the other 65% to 75% of the DNA that
has not been sequenced? These genes are clearly
inherited from our parents. It would not appear likely
that those genes would have been retained evolution-
arily along our ancestry if they performed no function.
Would not these regions be more likely to harbor the
differences that account for both survival genes and
familial patterns of disease inheritance? It is the non-
coding region of the DNA that is used to identify ones
parentage unequivocally. If one wants to prove that a
DNA sample is from a specific person, then the
sequences are determined for the highly variable non-
coding regions of the DNA. Those sequences not only
identify us as a specic person, but arguably it is those
sequences that uniquely dene us as a biological entity.
Logically, the fact that I am more like my father than
a gorilla is probably due to the fact that I inherited
those non-coding regions from my father and not a
gorilla (more-or-less...). Indeed, most of the DNA that
uniquely denes us is not within the human genome
sequence base. It would appear likely that the dis-
covery of relatively rare, single gene mutations (i.e.,
genetic defects) that result in altered proteins not crit-
ical for survival will be elucidated rst by the use of the
human gene bank data, and that complex polygenic
diseases will be more difficult to discover with this
dataset. Admittedly, there will also be important link-
ages which may co-segregate with those genes of inter-
est, so it will provide useful information to dene hot
spots of the genome that confer risk. Furthermore, the
gene sequences that are known do serve as impor-
tant islands of information and points of reference
within the vast and expansive sea of unknown DNA
code.
The logical consequences that follow from this line
of thinking should be raised from a scientific view-
point, even if controversial.
2. We probably have not completely broken the
genetic code. We understand the function of some of
the DNA sequences that code for proteins, but we are
only beginning to understand how promoter regions
define the transcriptome function. Messenger RNA
(mRNA) synthesis, mRNA splicing, and mRNA turnover
are ultimately regulated by DNA and RNA sequences
and by key interactions of the DNA and RNA with pro-
teins that often require metabolic alteration such as
phosphorylation, acetylation, or conformational shifts
via ligand binding with other molecules, including lipids.
For example, the open-reading frame gene sequences
for prostaglandin synthase or tumor necrosis factor
that translate into the primary amino acid sequences
for these proteins are virtually identical from person to
person, but the sequences upstream and downstream
of the coding region can differ signicantly from per-
son to person. Often these are single nucleotide poly-
morphisms or SNPs, which are inherited genes that
usually are not in encoding regions of the genome.
There are DNA sequences that provide docking bays
that allow proteins to bind and which function by
enhancing the transcription of the specic mRNA mol-
ecules that code for these proteins. These DNA regions
are called response elements. Among other mecha-
nisms, different SNPs can alter the binding of nuclear
transcriptional factors to these elements and modu-
late transcription levels. Thus, differences in these SNPs
may ultimately account for genetic differences in the
amount of mRNA of a critically important biological
molecule such as a cytokine or growth factor that reg-
ulates the host response to challenges and may result
in a phenotypic difference in host susceptibility or resis-
tance. Many genes have similar response element
sequences that enable them to be expressed or mod-
ulated in a concerted manner. In the future, the bio-
logical responsiveness and function of a molecule may
be predicted based upon the sequences of these reg-
ulatory elements that ank the open-reading frames.
The study of these regions will undoubtedly be made
much easier with the sequencing of the surrounding
coding regions, but this is a region likely to be where
true differences in hosts can be genetically identied
to enable personalized medicine approaches. Further-
more, it would appear likely that there are several other
kinds of genetic codes that determine how genes are
regulated that we have yet to discover.
3. The genes that make us different are also likely
to include those genes that explain the differences
among us in disease resistance and susceptibility. This
may seem to be an obvious point, but it is not a pop-
ular point of view. We tend to prefer the concept that
we are all genetically the same, or almost the same,
and that diseases are thrust upon us as a consequence
of environmental exposures, behaviors, or, even bet-
ter, external inuences that we cannot often control
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such as socioeconomic status, stress, or educational
attainment. However, the data are consistently telling
a different story. Studies of genetically identical twins
reared together or apart continue to provide compelling
scientic evidence that many major human illnesses
that affect the majority of the population ranging from
cardiovascular disease, to diabetes, to periodontal dis-
ease have a substantive genetic component.
1-4
Over
and over, studies tend to reach the same conclusion:
that about half of the variance in the expression of
health or disease can be explained by genes. This
means that half of the disease or the trait can, on aver-
age, be explained by the genes that we inherit from our
lineage and suggest that the remaining part is due to
environmental exposures or behaviors. Thus, this line
of thinking would suggest that in order to understand
the contribution of genes to risk, we will by necessity
need to better understand the role of those DNA
sequences that are beyond the coding regions. We will
need detailed SNP maps or haplotype analyses to
establish patterns of inheritance of multiple genes that
confer risk. Furthermore, we will need to better under-
stand the complexities of transcriptional biology as
well as translational and post-translational regulation
before we can appreciate the functional impact of these
genetic determinants on the disease phenotype.
In concluding this commentary, I would like to re-
emphasize that these points are raised as a caution-
ary and certainly not a negative note. For example,
genetic models of diseases have often used transgenic
or knock-out experimental approaches that over-
express or underexpress (or express dominant nega-
tive proteins) to determine the role of that specic mol-
ecule in disease expression or to create new models
of disease. Even if these models do not faithfully repro-
duce the human condition, they often lead to impor-
tant insights into mechanisms of pathogenesis. Stud-
ies that examine the role of regulatory genes using
these experimental approaches are in a minority, but
hold promise. Thus, important strides are being made.
However, admitting that there are large genetic differ-
ences between and among individuals (and differences
that we dont understand) is sometimes a more con-
troversial and difcult issue to grasp. However, using
differences in DNA sequences that are unique to the
individual or the individuals lineage to confer suscep-
tibility or resistance should not be viewed similarly to
using DNA evidence to identify who someones true
parents are (and that they are not a gorilla). Suggest-
ing that there are substantive, not minor, genetic dif-
ferences between individuals is not to suggest that
these differences (desirable or undesirable genes) fall
along ethno-racial, cultural, or socioeconomic group-
ings. However, it does suggest that reluctance to
explore these genetic differences will hamper a full
understanding of how gene-environment interactions
result in disease. In other words, the sooner we come
to grips with the realities and limitations of what we
know, and acknowledge that we are just at the very
beginning of this emerging eld of genomics, the more
likely we will not at some point in the future reminisce
with embarrassment at our forecasts of soon living in
Martian colonies with personalized full dental plans.
ACKNOWLEDGMENTS
At this 20th session of the ICPR, the organizers gave
a special award of recognition to Robert Singer, PhD.,
Senior Scientist at Procter & Gamble, in acknowledg-
ment of his scientic leadership and the impact he has
made in periodontal research and the biomedical sci-
entic community. This occasion was in honor of his
retirement from Procter & Gamble. His insight and
leadership within the industry have had a tremendous
impact over the past few decades in growing oral health
care from its historical roots in soap chemistry to a
growing age of biopharmaceuticals and biomedical
device applications. His work has extended beyond
basic research and corporate product development,
as he has been a strong and unfaltering champion for
basic and clinical periodontal research. His advocacy
and the sole support of Procter & Gamble have made
this ICPR conference a thriving tradition and an entity
that promotes new research, young investigators, and
the open exchange of ideas. Our committee and the
conference attendees would like to thank P&G for its
gracious support of the 20th ICPR and especially want
to acknowledge the outstanding periodontal research
contributions of Bob Singer, who is a true colleague and
friend.
I would also like to give my special thanks to my
co-chair, Dr. Ray Williams, Chair of Periodontology,
whose outstanding efforts were critical to the success
of the Conference and for personally assuring that it
was a well-organized, informative, pleasurable, and
memorable experience for all of us. Thanks also to the
UNC School of Dentistry (special thanks to Sharon
Grayden and Amy Williams, UNC Continuing Educa-
tion), the Department of Periodontology, the Compre-
hensive Center for Inflammatory Disorders, and the
UNC Center for Oral and Systemic Diseases. I want to
thank Jim Beck, Ray Williams, and Gary Armitage for
their thoughtful suggestions on this commentary.
REFERENCES
1. Michalowicz BS, Aeppli D, Virag JG, et al. Periodontal
ndings in adult twins. J Periodontol 1991;62:293-299.
2. Michalowicz BS, Aeppli DP, Kuba RK, et al. A twin study
of genetic variation in proportional radiographic alveolar
bone height. J Dent Res 1991;70:1431-1435.
3. Michalowicz BS, Wolff LF, Klump D, et al. Periodontal
bacteria in adult twins. J Periodontol 1999;70:263-273.
4. Michalowicz BS, Diehl SR, Gunsolley JC, et al. Evidence
of a substantial genetic basis for risk of adult periodonti-
tis. J Periodontol 2000;71:1699-1707.
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