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Discussion

Genetic Susceptibility to Keloid Disease and HypertrophicScarring: Transforming Growth Factor 1 CommonPolymorphisms and Plasma Levels

Discussion by George P. Yang, M.D., Ph.D., Jen-Tsan Ashley Chi, M.D., Ph.D., andMichael T. Longaker, M.D.

In their article, Bayat et al. present an at-tempt to link a genetic predisposition to scar-ring with known single nucleotide polymor-phisms in the transforming growth factor 1(TGF-1) gene. As evidenced by their negativefindings, the ability to find genetic markers forpatients predisposed to keloid or hypertrophicscar formation has been an elusive target forinvestigators.

Benign conditions such as keloid and hyper-trophic scar formation remain among the mostformidable disorders to study at the molecularlevel. These indolent processes often take de-cades to reach a clinically significant point.1,2

In addition, they are multifactorial in origin,

and it may be that only some of these factorsneed be present for the condition to progress.In the case of keloids, such factors may beenvironmental rather than genetic, as demon-strated by the clinical observation of their ten-dency to form in areas of the skin that aresubject to increased physical stress.1,2 Thus, theinvestigator is forced to examine a number ofvariables that may not be easily brought to-gether in a single model.

TGF-1 has been intensely studied in thepathogenesis of excessive scarring because of

its known biological activities, including stimu-lation of fibroblast proliferation and deposi-tion of extracellular matrix. Given the patho-logic findings in keloid formation of athickened dermis with increased extracellularmatrix deposition, it is clear why so much efforthas been directed toward examining the roleof TGF-1 in excessive scarring conditions. Ad-ditional evidence has shown that overexpres-sion of Smad2 in the epidermis of transgenic

mice can lead to abnormally thickened dermis,further implicating the TGF signaling path-way as important in excessive scarring.3 Fur-thermore, studies of TGF signaling in sclero-derma, another fibrotic disorder in whichexcessive TGF activity has been implicated,point to deficiencies in the normal negativefeedback signal of Smad7 as a potential molec-ular defect in the disease.4

Our laboratory has also directed a great dealof attention at the role of TGF-1 and its iso-forms in keloid pathogenesis. However, afteryears of focused research on this growth factorand components of its signaling pathway, we(like Bayat and coworkers) are still unable to

clearly define a molecular defect that directlylinks the presence of excessive TGF activityand excessive scarring. For this reason, we be-lieve that the future of research into keloidpathogenesis may benefit tremendously fromnewly emerging techniques for high-through-put screening, as exemplified by cDNAmicroarray.

The epidemiology of keloid formationclearly suggests a genetic component, as evi-denced by the 3 percent of patients who seemto have a hereditary form of keloid disease.5

However, keloid formation is an indolent, be-nign process, suggesting that the alterationspresent at the molecular level would be smallin comparison with the large-scale disturbancesseen in rapidly growing forms of cancer. Thus,a highly sensitive methodology is required todetect these differences. Furthermore, exami-nation of the known biology of hypertension,another benign process with an apparent ge-netic predisposition, shows the same difficul-

Received for publication May 20, 2002.

DOI: 10.1097/01.PRS.0000041537.30632.04

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ties in determining the precise molecularmechanisms in the disease.

Like keloid formation, essential hyperten-sion is a heavily studied benign condition witha strong genetic component, as suggested byepidemiologic studies.6,7 Despite intensive ex-

amination, the molecular mechanisms leadingto essential hypertension remain nearly as un-clear as for keloid formation. This highlightswhat we believe to be a feature of most benigndiseases: they are multifactorial in origin. Someof these factors may be environmental ratherthan genetic and might not have a singleclearly identifiable molecular trigger.

Performing a single, or candidate, gene ex-pression analysis in studying these conditions islikely to be an exercise in frustration. Occa-sionally, a fortuitous happenstance for the in-

vestigator might lead to unique insight into abenign condition. More often, the investigatoris faced with conflicting data and no clear an-swer. The ability to survey gene expression inthousands of genes at a given time, as providedby cDNA microarray, offers clear advantageswhen examining these benign conditions.

First, this approach allows the investigationof multiple related genes in a single analysis.Second, inclusion of the entire human genomein the analysis, as is now possible with currenttechnology, allows the investigator to identify

previously unanticipated relationships and toprovide new insights into mechanisms of dis-ease.8,9 Finally, the availability of powerful soft-ware to analyze the data through clusteringanalysis will allow identification of a number ofgenes that might be associated with keloid for-mation. It may be the case, for example, thatwithin a cluster of potential candidate genes,aberrant expression of four of five genes asso-ciated with keloids may be required to be pre-dictive. Microarray analyses will have the powerto help us make that determination.

Bayat et al. chose a single-gene approach toexamine a potential relationship between sin-gle nucleotide polymorphisms and keloid for-mation. Performing this analysis for one geneis already a significant amount of work. Oligo-nucleotide arrays allow screening for the pres-ence of multiple single nucleotide polymor-phisms in multiple genes in a high-throughputand parallel fashion.10 As Bayat et al. noted intheir conclusions, they are currently lookingfor novel polymorphisms in TGF-1 and othermembers of the TGF signaling pathway. Thiscan be rapidly accomplished using microarray

analysis, which may be a very powerful tool forthe analysis of known pedigrees of familial ke-loid formation. In addition, rapid progress inthe field of bioinformatics will enable scientiststo make sense of the enormous amount of datathus generated.

The original cDNA microarrays developedby the laboratory of Pat Brown at StanfordUniversity were designed to examine differ-ences in expression patterns, in contrast to theuse of microarray to perform genotyping asdescribed above.11 One novel approach to theanalysis of the microarray data provided byadvances in bioinformatics that has proved verypowerful is the use of clustering software,which is especially relevant in the study of mul-tifactorial disease. Often there is not a singledefect that is present in every patient; however,

there is often a group of genes that demon-strate abnormal regulation. Identification ofthese aberrations is possible with the use ofdifferent analytic and statistical programs toidentify the disease patterns of gene expres-sion or single nucleotide polymorphisms. It ispossible that a patient who is predisposed toforming keloids may be identified by a ge-nome-wide microarray, followed by clustering,to see if they share a pattern of gene expressionsimilar to other such patients.

Despite its power, cDNA microarray analysis

is fraught with potentially confounding vari-ables. Microarrays are sensitive enough to de-tect fairly small differences in gene expressionfrom one sample to another, including differ-entiating male versus female specimens basedon sex-specific gene expression, which meansthat the investigator requires very strict con-trols to generate differences that can be believ-able. The vast amount of data generated alsorequires a sophisticated software package toidentify true differences and associations.

Once a microarray analysis has been per-formed, the investigator may still be left with alarge number of genes that must be investi-gated. It is important that potentially impor-tant genes be analyzed to correlate their func-tion with the actual biology. A critical error inmicroarray analysis is to assume that a genethat is differentially regulated must, therefore,be important in the biology studied. It is stillnecessary to perform the experiments to de-fine the role of the gene in the disease processto demonstrate its relevance.

The advent of cDNA microarray also doesnot mean that the single-gene approach to

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analysis of a benign disorder is not withoutmerit. In many instances, as with keloid forma-tion, a significant body of knowledge impli-cates at least one gene in the disease process.In some cases, that gene may represent amarker of the disease condition, or it may play

a significant role in disease progression whilenot being a universal mechanism.

Despite our interest in the use of cDNA mi-croarray in the analysis of keloid formation, wedo not believe that this technology will providethe final answers. Rather, we hope that mi-croarray analysis will provide us with fresh in-sights into additional genes not previously im-plicated that may be involved in the formationof keloid disease. These genes will be thesource of new testable hypotheses about themolecular pathogenesis of keloid formation.

Michael T. Longaker, M.D.Childrens Surgical Research ProgramDepartment of SurgeryStanford University Medical School257 Campus DriveStanford, Calif. 94305-5148longaker@stanford.edu

REFERENCES

1. Murray, J. C. Scars and keloids. Dermatol. Clin. 11: 697,1993.

2. Ramakrishnan, K. M., Thomas, K. P., and Sundararajan,C. R. Study of 1,000 patients with keloids in SouthIndia. Plast. Reconstr. Surg. 53: 276, 1974.

3. Ito, Y.,Sarkar, P.,Mi, Q.,et al. Overexpression of Smad2reveals its concerted action with Smad4 in regulatingTGF-beta-mediated epidermal homeostasis. Dev. Biol.236: 181, 2001.

4. Dong, C., Zhu, S., Wang, T., et al. Deficient Smad7expression: A putative molecular defect in sclero-derma. Proc. Natl. Acad. Sci. U.S.A. 99: 3908, 2002.

5. Marneros, A. G., Norris, J. E., Olsen, B. R., and Reichen-berger, E. Clinical genetics of familial keloids. Arch.

Dermatol. 137: 1429, 2001.6. Sharma, P., Fatibene, J., Ferraro, F., et al. A genome-

wide search for susceptibility loci to human essentialhypertension. Hypertension 35: 1291, 2000.

7. Timberlake, D. S., OConnor, D. T., and Parmer, R. J.Molecular genetics of essential hypertension: Recentresults and emerging strategies. Curr. Opin. Nephrol.Hypertension10: 71, 2001.

8. Lander, E. S. Array of hope. Nat. Genet. 21(1 Suppl.): 3,1999.

9. Schena, M., Heller, R. A., Theriault, T. P., et al. Mi-croarrays: Biotechnologys discovery platform forfunctional genomics [see comments]. Trends Biotech-nol. 16: 301, 1998.

10. Hacia, J. G., Fan, J. B., Ryder, O., et al. Determinationof ancestral alleles for human single-nucleotide poly-morphisms using high-density oligonucleotide arrays.Nat. Genet. 22: 164, 1999.

11. Schena, M., Shalon, D., Davis, R. W., and Brown, P. O.Quantitative monitoring of gene expression patternswith a complementary DNA microarray [see com-ments]. Science 270: 467, 1995.

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