periodontal research: molecular determinants of risk

8/12/2019 Periodontal research: Molecular determinants of risk

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Ann Periodontol

Reflections on the 20th International Conference on

Periodontal Research: Molecular Determinants of RiskWith 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.

CONFERENCE OVERVIEW

The 20th ICPR was one of the first periodontalresearch conferences of the post-genomic era. It wasorganized with the purpose of providing periodontalscientists with an overview of what the future may hold

using microbial and human genomics as tools toexplore pathogenesis and redefine diagnosis, preven-tion, and treatment. It brought experts in moleculargenomics 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 explorechanges in transcriptome expression in cells, tissues,and animal models. Elegant techniques were shownthat begin to fully characterize the biodiversity of theoral biofilm and to characterize the virulence genes oforganisms with genomic approaches. The goal of theconference 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. Theintent was to begin the process of identifying specificgenetic markers that confer risk or resistance, and cer-tain microbial virulence genes that are critical to patho-genesis to reconstruct definitions of diseases that arenot necessarily generalizable to the population at large,but provide a detailed, specific characterization at apatient level or within a smaller subset of individuals.Papers were presented that provided new insights intothe thought processes that will be needed to establishnew clusters of clinical and laboratory findings to defineperiodontal disease syndromes and to provide new

definitions of disease that better characterize the oralcondition as it serves as an exposure for systemiccomplications. Commonalities of pathogenesis wereraised when periodontal disease was considered as atriad of clinical signs, oral infection, and inflammatoryresponse rather than a single cluster of clinical find-ings. These mechanisms extend from local tissuedestruction to pathways that involve the vasculatureand pregnancy. Papers on the influence of lipid andcarbohydrate metabolism, as well as obesity and dia-betes as metabolic conditions, were discussed as tohow they modify periodontal disease expression orserve as systemic stressors. Discussions ranged from

lipid modulation of inflammation to discussions of thedifferences between anthropomorphic definitions ofcentral obesity, body mass index, and waist-to-hip ratioon disease models. These papers and discussionshelped to clarify how obesity and diabetes serve as

effect modifiers as one examines the role of oral infec-tion on periodontal status and systemic complicationssuch as cardiovascular disease.

The research trend manifested by this ICPR meet-ing is clear. We are rapidly moving into an era whenmolecular laboratory findings, especially protein orDNA-based, using host or pathogen biological sampleswill be tightly integrated into our traditional clinicaldefinitions of disease. It is the promise of the futurethat we will eventually be able to better define 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 thenbe able to offer a specific preventive or therapeuticstrategy that is customized to the individual. This Con-ference was a successful step towards describing theseapproaches to explore this formidable challenge ofpersonalized medicine. And, as a consequence ofreflection and debate on this transitional thinking andin the spirit of the ICPR, which traditionally fosters anopen-minded exchange of controversial ideas, the fol-lowing personal commentary on this post-genomicfuture is offered.

COMMENTARY

Many of us in science can remember the excitementgenerated by the original scientific breakthrough ofWatson and Crick that started it all 50 years ago andcan 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 ofrelevant oral pathogens. It is a phenomenal time to bein science, as these shared national scientific land-marks of achievement rival the collective effort ofputting a manned spacecraft on the moon. These newgenetic roadmaps should help us navigate our exper-imental pathways as we try to decipher the complexintricacies of microbial virulence and pathogenesis,


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Reflections 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 informationhas birthed its own discipline (bioinformatics) and ispurported to eventually enable us to achieve the com-

mon goal of personalized medicine that includes den-tistry. That is, the prevention and treatment of humanailments will be repackaged not to deliver what gen-erally works best and is globally safe for everyone, butrather, in the context of optimizing the prevention andintervention specifically for the unique patient usinginformation 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 dentalresearch, 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 tounfold.

Inevitably, changing trends in research are precipi-tated by new discovery and the creation of new tools.Landmark technological advances that create newmethods can sometimes create self-validating scien-tific lines of investigation rather than simply providebetter tools for scientific discovery. Thus, as we moveinto this new area of genomics, one is reminded of theafterglow of post-Apollo landing times when we glee-fully discussed living on Mars, just as today with thesequencing of the human genome some now forecastthe imminent end of human suffering from disease. At

times, this optimism is fueled by the ecumenical mediahype for one trendy research method or another. Butthe history of biomedical research is spotted with exam-ples of ineffective lines of investigation, suggesting thatsometimes the tools we use drive the scientific ques-tions we pose rather than the other way around. Forexample, with the perfection of electron microscopy,structural biology became the science of the day, andthen there was the era of monoclonal antibodies. Howmany research facilities still harbor antigen-specificmonoclonal cell lines that may have been of interest butwere never characterized? With the explosion in mol-ecular biology over the last 2 decades, it seems that

virtually every scientific discipline has now beenrenamed to include the word molecular. And in recentyears, how many laboratories have studied a uniquegene that has been cloned, sequenced, and, via trans-genic models, been over- or underexpressed, and stillremains without a clear function?

With the sequencing of the human genome and agrowing list of microbial genomes providing the criti-cal blueprints for potential experiments, some specificthoughts came to mind. I would like to briefly raisesome 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 severaldecades. 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 first 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 aretranscribed into messenger RNA and then translatedinto proteins. That represents only about 25% to 35%of the entire DNA sequence. There are huge areas ofDNA in between these open-reading frames that arethought to play some regulatory and structural role formaintaining the genome. The data in the humangenome map contain, for example, the entire sequencefor type I collagen alpha chain. It is often popular tocite in the literature that the differences between thegenes for man and a gorilla differ by less than 5%. It

is no surprise that the sequence of type I collagen forman is almost identical to that of a gorilla. It has beenargued that the differences between gorillas and mencan be explained by that infinitesimally small, butsomehow magically transforming, 5% that representsthe 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, collagenis a basic building block of connective tissue and itwould be inefficient for us not to have utilized this pro-tein as an evolutionarily conserved molecule. Clearly,

genes that code for proteins representing the commonbuilding blocks of life that are needed for a biologi-cally viable mammal should logically be quite similar.These sequences are conserved because mothernature found them to be useful so they need not bereinvented. But what about those unknown sequencescontained within the non-coding DNA? Many yearsago these genes were termed nonsense genes, as thegene products did not result in protein sequences andthere is tremendous diversity in these sequences. It isthe unique combination of sequences in this region ofthe total DNA that is used to incriminate a suspectedmurderer, for example. There are regions of DNA that

contain so-called random repeat areas of DNA thatdo not code for protein but can be used to identify aperson uniquely and establish parentage and familiallinkages. However, it might be argued that it wouldalso seem totally evolutionarily inefficient that any DNAsequence 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 thesesequences are not, in fact, random and contain infor-mation as yet to be discovered. These are thesequences that we inherited from our parents that giveus our special personalities and physical features andthey are probably no more random than those regions

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that encode for protein that we also considered to berandom gibberish just 50 years ago. One might recallthat prior to Watson and Crick, the entire 4-basesequence of the entire chromosomal DNA was alsoconsidered a random chain of bases. We now knowthat it is clearly not random and that it has a critical

importance with regard to lineage and heritance. Thiswould suggest that there is much more of the geneticcode yet to be unraveled.

The limited nature of the DNA sequence decodingdata 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 personuniquely, it is not done by comparing DNA from anindividual to the human genome sequence data. Afterall, using just these data, we could not really distinguishvery efficiently 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 especiallymost 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 thathas not been sequenced? These genes are clearlyinherited from our parents. It would not appear likelythat 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 thedifferences that account for both survival genes andfamilial 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 aDNA sample is from a specific person, then thesequences are determined for the highly variable non-coding regions of the DNA. Those sequences not onlyidentify us as a specific person, but arguably it is thosesequences that uniquely define us as a biological entity.Logically, the fact that I am more like my father thana gorilla is probably due to the fact that I inheritedthose non-coding regions from my father and not agorilla (more-or-less...). Indeed, most of the DNA thatuniquely defines us is not within the human genomesequence 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 first by the use of thehuman gene bank data, and that complex polygenicdiseases will be more difficult to discover with thisdataset. 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 define hotspots of the genome that confer risk. Furthermore, thegene sequences that are known do serve as impor-tant islands of information and points of referencewithin the vast and expansive sea of unknown DNAcode.

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 thegenetic code. We understand the function of some ofthe DNA sequences that code for proteins, but we areonly beginning to understand how promoter regions

define the transcriptome function. Messenger RNA(mRNA) synthesis, mRNA splicing, and mRNA turnoverare ultimately regulated by DNA and RNA sequencesand by key interactions of the DNA and RNA with pro-teins that often require metabolic alteration such asphosphorylation, acetylation, or conformational shiftsvia ligand binding with other molecules, including lipids.For example, the open-reading frame gene sequencesfor prostaglandin synthase or tumor necrosis factorthat translate into the primary amino acid sequencesfor these proteins are virtually identical from person toperson, but the sequences upstream and downstreamof the coding region can differ significantly from per-

son to person. Often these are single nucleotide poly-morphisms or SNPs, which are inherited genes thatusually are not in encoding regions of the genome.There are DNA sequences that provide docking baysthat allow proteins to bind and which function byenhancing the transcription of the specific mRNA mol-ecules that code for these proteins. These DNA regionsare called response elements. Among other mecha-nisms, different SNPs can alter the binding of nucleartranscriptional factors to these elements and modu-late transcription levels. Thus, differences in these SNPsmay ultimately account for genetic differences in theamount 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 resultin a phenotypic difference in host susceptibility or resis-tance. Many genes have similar response elementsequences 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 maybe predicted based upon the sequences of these reg-ulatory elements that flank the open-reading frames.The study of these regions will undoubtedly be mademuch easier with the sequencing of the surroundingcoding regions, but this is a region likely to be wheretrue differences in hosts can be genetically identified

to enable personalized medicine approaches. Further-more, it would appear likely that there are several otherkinds of genetic codes that determine how genes areregulated that we have yet to discover.

3. The genes that make us different are also likelyto include those genes that explain the differencesamong us in disease resistance and susceptibility. Thismay seem to be an obvious point, but it is not a pop-ular point of view. We tend to prefer the concept thatwe are all genetically the same, or almost the same,and that diseases are thrust upon us as a consequenceof environmental exposures, behaviors, or, even bet-ter, external influences that we cannot often control


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Reflections on the 20th International Conference on Periodontal Research Volume 7 Number 1 December 2002

such as socioeconomic status, stress, or educationalattainment. However, the data are consistently tellinga different story. Studies of genetically identical twinsreared together or apart continue to provide compellingscientific evidence that many major human illnessesthat affect the majority of the population ranging from

cardiovascular disease, to diabetes, to periodontal dis-ease have a substantive genetic component.1-4 Overand over, studies tend to reach the same conclusion:that about half of the variance in the expression ofhealth or disease can be explained by genes. Thismeans that half of the disease or the trait can, on aver-age, be explained by the genes that we inherit from ourlineage and suggest that the remaining part is due toenvironmental exposures or behaviors. Thus, this lineof thinking would suggest that in order to understandthe contribution of genes to risk, we will by necessityneed to better understand the role of those DNAsequences that are beyond the coding regions. We will

need detailed SNP maps or haplotype analyses toestablish patterns of inheritance of multiple genes thatconfer risk. Furthermore, we will need to better under-stand the complexities of transcriptional biology aswell as translational and post-translational regulationbefore we can appreciate the functional impact of thesegenetic 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 transgenicor knock-out experimental approaches that over-express or underexpress (or express dominant nega-

tive proteins) to determine the role of that specific mol-ecule in disease expression or to create new modelsof 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 usingthese experimental approaches are in a minority, buthold promise. Thus, important strides are being made.However, admitting that there are large genetic differ-ences between and among individuals (and differencesthat we dont understand) is sometimes a more con-troversial and difficult issue to grasp. However, usingdifferences in DNA sequences that are unique to the

individual or the individuals lineage to confer suscep-tibility or resistance should not be viewed similarly tousing DNA evidence to identify who someones trueparents 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 thatthese differences (desirable or undesirable genes) fallalong ethno-racial, cultural, or socioeconomic group-ings. However, it does suggest that reluctance toexplore these genetic differences will hamper a fullunderstanding of how gene-environment interactions

result in disease. In other words, the sooner we cometo grips with the realities and limitations of what weknow, and acknowledge that we are just at the verybeginning of this emerging field of genomics, the morelikely we will not at some point in the future reminiscewith 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 gavea special award of recognition to Robert Singer, PhD.,Senior Scientist at Procter & Gamble, in acknowledg-ment of his scientific leadership and the impact he hasmade in periodontal research and the biomedical sci-entific community. This occasion was in honor of hisretirement from Procter & Gamble. His insight andleadership within the industry have had a tremendousimpact over the past few decades in growing oral healthcare from its historical roots in soap chemistry to agrowing age of biopharmaceuticals and biomedicaldevice applications. His work has extended beyondbasic research and corporate product development,as he has been a strong and unfaltering champion forbasic and clinical periodontal research. His advocacyand the sole support of Procter & Gamble have madethis ICPR conference a thriving tradition and an entitythat promotes new research, young investigators, andthe open exchange of ideas. Our committee and theconference attendees would like to thank P&G for itsgracious support of the 20th ICPR and especially wantto acknowledge the outstanding periodontal researchcontributions of Bob Singer, who is a true colleague andfriend.

I would also like to give my special thanks to myco-chair, Dr. Ray Williams, Chair of Periodontology,whose outstanding efforts were critical to the successof the Conference and for personally assuring that itwas a well-organized, informative, pleasurable, andmemorable experience for all of us. Thanks also to theUNC School of Dentistry (special thanks to SharonGrayden and Amy Williams, UNC Continuing Educa-tion), the Department of Periodontology, the Compre-hensive Center for Inflammatory Disorders, and theUNC Center for Oral and Systemic Diseases. I want tothank Jim Beck, Ray Williams, and Gary Armitage fortheir thoughtful suggestions on this commentary.

REFERENCES

1. Michalowicz BS, Aeppli D, Virag JG, et al. Periodontalfindings in adult twins. J Periodontol1991;62:293-299.

2. Michalowicz BS, Aeppli DP, Kuba RK, et al. A twin studyof genetic variation in proportional radiographic alveolarbone height. J Dent Res1991;70:1431-1435.

3. Michalowicz BS, Wolff LF, Klump D, et al. Periodontalbacteria in adult twins. J Periodontol1999;70:263-273.

4. Michalowicz BS, Diehl SR, Gunsolley JC, et al. Evidenceof a substantial genetic basis for risk of adult periodonti-tis. J Periodontol2000;71:1699-1707.

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