Abstract
Objective
Hypertrophic scars (HTS) occur in 30–72% patients following thermal injury. Risk factors include skin color, female gender, young age, burn site, & burn severity. Recent correlations between genetic variations and clinical conditions suggest that single nucleotide polymorphisms (SNPs) may be associated with HTS formation. We hypothesized that a SNP in the p27kip1 gene (rs36228499) previously associated with decreased restenosis after coronary stenting would be associated with lower Vancouver scar scale (VSS) measurements and decreased itching.
Methods
Patient & injury characteristics were collected from adults with thermal burns. VSS scores were calculated at 4–9 months following injury. Genotyping was performed using real time PCR. Logistic regression was used to determine risk factors for hypertrophic scar as measured by a VSS score >7.
Results
300 subjects had a median age of 39 years (range 18–91); 69% were male & median burn size was 7% TBSA (range 0.25–80). Consistent with literature, the p27kip1 variant SNP had an allele frequency of 40%, but was not associated with reduced HTS formation or lower itch scores in any genetic model. HTS formation was associated with American Indian/Alaskan Native race (OR, 12.2; P=0.02), facial burns (OR, 9.4; P=0.04), and burn size ≥20% TBSA (OR, 1.99; P=0.03).
Conclusions
Whereas the p27kip1 SNP may protect against vascular fibroproliferation, the effect cannot be generalized to cutaneous scars. Our study suggests that American Indian/Alaskan Native race, facial burns, and higher %TBSA are independent risk factors for HTS. The American Indian/Alaskan Native association suggests that there are potentially yet-to-be-identified genetic variants.
Keywords: single nucleotide polymorphism, genetic variation, hypertrophic scar
INTRODUCTION
Whereas advances in the treatment of burn injuries have led to decreases in both morbidity and mortality, hypertrophic scars (HTS) following burn injury remain a significant long-term functional and aesthetic problem. Prevalence of hypertrophic scarring in the literature ranges from 32–72%.1 These raised, red, and pruritic scars lead to decreases in quality of life scores.2 Patients complain of pain, pruritus, and decreased function as a result of these scars. Identified risk factors for hypertrophic scar development include young age, female gender, dark skin, neck or upper limb burns, multiple surgical procedures, greater than 3 weeks to healing, meshed skin graft use, and burn severity.1
Recent correlations between genetic variations and clinical conditions or outcomes3–5 raise questions as to whether single nucleotide polymorphisms (SNPs) are associated with HTS development. A study by van Tiel and colleagues found that a SNP in the p27kip1 gene, −838C>A, (rs36228499) was associated with a decreased risk of in-stent restenosis of cardiac stents. Subjects who were homozygous for the variant allele, A, had a decreased risk of in-stent restenosis which corresponded with enhanced promoter activity of the variant allele. P27kip1 is expressed in the healing wounds in rats,6 has been found to control cytokinesis in murine fibroblasts,7 and administration of p27kip1 reportedly decreased epithelial proliferation following glaucoma filtration surgery in rabbits.8 A fibroproliferative response is suspected in the pathogenesis of both vascular stenosis and HTS. Therefore, we hypothesized that p27kip1 −838A>C would be associated with lower scores on the Vancouver Scar Scale (VSS) and lower itch scores.
METHODS
Patient recruitment and Data Collection
Adult patients (≥18 years of age) who were deemed at–risk for developing hypertrophic scar based on time to healing and depth of wounds were recruited for enrollment. Both Institutional Review Board approval and a Federal Certificate of Confidentiality were obtained for this project.
A whole blood sample from each subject was collected. Patient characteristics and outcome data were collected from the electronic medical record. Race and ethnicity data are reported in accordance with the NIH policy.9 Subjects were seen at two follow up visits; one at 1–5 months following injury and one at 6–12 months following injury. At these visits the subject’s wounds were assessed by a research nurse who was blinded to the subject’s genotype and their VSS scores and itch scores were recorded. An example of the VSS is seen in Table 1. Itch scores were derived from a patient’s self-report on how badly their scar itched on a scale of 0 to 10 with zero being no itch at all.
Table 1.
Vancouver Scar Scale (0–13)
| Pigmentation (0–2) | Normal | 0 |
| Hypo pigmentation | 1 | |
| Hyperpigmentation | 2 | |
| Vascularity (0–3) | Normal | 0 |
| Pink | 1 | |
| Red | 2 | |
| Purple | 3 | |
| Pliability (0–5) | Normal | 0 |
| Supple | 1 | |
| Yielding | 2 | |
| Firm | 3 | |
| Banding | 4 | |
| Contracture | 5 | |
| Height (0–3) | Normal (flat) | 0 |
| 0–2mm high | 1 | |
| 2–5mm high | 2 | |
| >5mm high | 3 |
Genotyping
Genotypes for p27kip1-838C>A SNP (rs36228499) were determined by allelic discrimination assay using TaqMan®-based real time PCR (RT-PCR) as previously described.10,11 Briefly, 5–20ng of DNA were amplified using Custom TaqMan® SNP Assay with the Applied Biosystems 7900HT Real Time PCR system under standard conditions. A unique pair of fluorescent dyes, Minor Groove Binder (MGB) probes, were used. The fluorescence of each well was recorded and analyzed with SDS software (Applied Biosystems), an allelic discrimination plot was generated, and calls were assigned.
Statistical Analysis
Continuous data are presented as medians and ranges and were compared using Student’s t-test or ANOVA. Categorical data are presented as counts and percentages and were compared using χ2 analysis. For each comparison, exact P values are reported and considered significant if P<0.05. To determine deviations from Hardy-Weinberg equilibrium, we compared observed genotype frequencies to expected frequencies.
The primary outcome was development of HTS as measured by a VSS score of >7. The secondary outcome measure was clinically significant itch as measured by an itch score of >4. We used the itch score as a surrogate marker for HTS as pruritus is a common clinical complaint of these scars. The highest VSS and itch scores of the two follow up assessments were used for analysis. Multiple logistic regression analysis was used to test for associations between genotype and HTS development. We tested additive, co-dominant and recessive models correcting for age, gender, race/ethnicity, percent total body surface area (TBSA) burn, and burn location. Data were analyzed using Stata 12 Statistical Software (Stata Corp, LP. College Station, TX). A power analysis was performed using PS: Power and Sample Size Calculations version 3.0.43.12
RESULTS
Patient Characteristics and Clinical Outcomes
Three hundred subjects who completed a prospective observational study to correlate SNPs with HTS development were analyzed. Median age was 39 years (range 18–91), 206 (69%) subjects were male and 235 (79%) were Caucasian. Median percent TBSA was 7.1 (range 0.25–80). Patient characteristics are summarized in Table 2. On average, VSS scores were higher at the first follow-up assessment (6.1±2.8 vs. 3.8±3.5; P<0.001). Using the worst score from two follow up assessments, the average total VSS score was 7±2.2. The worst average itch score was 3.7±2.7. Table 3 summarizes the scar outcomes. A power analysis, performed post-hoc, revealed a power of 0.94 with an α of 0.05 to detect a difference of 1 on the VSS with a standard deviation of ±2.
Table 2.
Subject Characteristics (N=300)
| n (%) | |
|---|---|
| Age, median (range) | 39 (18–91) |
| Male Gender | 206 (69) |
| Race/Ethnicity | |
| White | 235 (79) |
| American Indian/Alaskan Native | 9 (3) |
| Asian | 19 (6) |
| Black/African American | 11 (3.7) |
| Native Hawaiian/Pacific Islander | 1 (0.3) |
| Hispanic | 24 (8) |
| % TBSA, median (range) | 7.1 (0.25–80) |
Table 3.
Scar Outcomes
| mean ± SD |
|
|---|---|
| Worst Total Follow-up score on VSS | 7 ± 2.2 |
| Worst Follow-up Pigmentation Score | 1.8 ± 0.5 |
| Worst Follow-up Vascularity Score | 2.1 ± 0.8 |
| Worst Follow-up Pliability Score | 2.2 ± 1.1 |
| Worst Follow-up Height Score | 1.1 ± 0.7 |
| Worst Follow-up Itch Score | 3.7 ± 2.7 |
Associations of Clinical Factors with Vancouver Scar Scale and Itch Scores
We looked at a number of clinical variables to determine if there were any associations with VSS or itch scores. One hundred and twenty six subjects (42%) had a VSS score greater than 7. One hundred and forty two subjects (47%) reported an itch score of greater than 4.
Children have a higher prevalence of hypertrophic scarring compared to adults;13 therefore, we divided our cohort into three age groups (18–21 yrs, 22–64 yrs, >65yrs) to determine if younger age is associated with higher VSS or itch scores (Table 4). There was no difference in the average VSS score between the three age groups. However, we did see a statistically significant difference in itch scores with subjects ≥65 years of age reporting lower itch scores (P=0.04). There were no differences in average VSS or itch scores between males and females (Table 4, VSS: 7.1±2.3 vs. 7±2, P=0.88; itch: 3.5±2.6 vs. 4.1±3, P=0.16).
Table 4.
Vancouver Scar Scale (VSS) or Itch Scores and Associations with Subject and Injury Characteristics
| VSS Score* |
Itch Score* |
||
|---|---|---|---|
| Age 18–21 (n=31) | 7.4±2.3 | 3.9±2.6 | |
| Age 22–64 (n=241) | 7±2.2 | 3.8±2.7 | |
| Age ≥ 65 (n=28) | 6.6±2.4 | 2.5±2.7 | |
| P-value† | 0.46 | 0.04 | |
| Male (n=206) | 7.1 ± 2.3 | 3.5 ± 2.6 | |
| Female (n=93) | 7 ± 2 | 4.1 ± 3 | |
| P-value† | 0.88 | 0.16 | |
| Race/Ethnicity | |||
| White (n=235) | 6.9±2.2 | 3.5±2.6 | |
| American Indian/Alaskan Native (n=9) | 9±1.6 | 4.1±2.4 | |
| Asian (n=19) | 8.1±1.9 | 5.1±3.1 | |
| Black/African American (n=11) | 7.9±2.5 | 5.3±3.5 | |
| Native Hawaiian/Pacific Islander (n=1) | 8 | 0 | |
| Hispanic (n=24) | 6.75±2.3 | 3.9±2.8 | |
| P-value† | 0.01 | 0.08 | |
| Face (n=8) | 8.4±1.1 | 6.1±2.1 | |
| Torso (n=48) | 4.9±3.1 | 4.9±3.1 | |
| Upper Extremity (n=108) | 3.3±2.6 | 3.3±2.6 | |
| Lower Extremity (n=103) | 3.3±2.4 | 3.3±2.4 | |
| P-value† | 0.01 | <0.001 | |
| TBSA<20% (n=230) | 6.8±2.2 | 3.4±2.7 | |
| TBSA≥ 20% (n=69) | 7.7±2.1 | 4.6±2.7 | |
| P-value† | 0.002 | 0.002 | |
mean± standard deviation
P-value derived from parametric ANOVA for VSS and non-parametric ANOVA for itch score
Using the NIH race/ethnicity categories; 235 subjects were Caucasian (78%), 9 subjects were American Indian or Alaskan Native (3%), 19 were Asian (6%), 11 were Black or African American (3.7%), 1 subject was Native Hawaiian or Pacific Islander (0.3%) and 24 subjects were Hispanic (8%). Average VSS and itch scores by race/ethnicity are summarized in Table 4. There was a statistically significant difference in average VSS scores by race/ethnicity (P=0.01); in a post-hoc Sidak pairwise comparison this difference appeared between the Caucasian and American Indian/Alaskan Native subjects (P=0.057). There was a trend toward higher itch scores in the American Indian/Alaskan Native, Asian, and Black/African American subjects, however, this difference did not reach statistical significance (P=0.08).
Results of the average VSS and itch scores by burn location are summarized in Table 4. Compared to other body sites, facial burns had higher VSS scores (P=0.01) and higher itch scores (P<0.001). Lastly, we looked at percent TBSA and average VSS and itch scores. We divided the cohort based on TBSA<20% and TBSA≥20%. For subjects with ≥20% TBSA burns, the average VSS score was 7.7±2.1 which was significantly higher than those with <20% TBSA (6.8±2.2, P=0.002; Table 4). Similarly, subjects with ≥20% TBSA burns had significantly higher itch scores compared to those with <20% TBSA burns (4.6±2.7 vs. 3.4±2.7; P=0.002).
P27kip1 SNP is Not Associated with Higher VSS Scores
Ninety-eight subjects were homozygotes for the common variant (CC). One hundred and thirty-nine subjects were heterozygotes (CA). Sixty-three subjects were homozygotes for the rare variant of interest (AA). The A variant allele frequency in our population was 44%, which is consistent with the previously published literature; our population was in Hardy Weinberg equilibrium. We evaluated patient characteristics by p27kip1 genotype and saw no difference between the genotypes and any patient characteristic (Table 5). Also, there is no difference in the average total VSS score, the VSS subcategory scores, or average itch scores between genotypes (Table 6).
Table 5.
Subjects Characteristics by p27kip1 Genotype
| Total (N=300) |
CC (n=98, 33%) |
CA (n=139, 46%) |
AA (n=63, 21%) |
P-value | |
|---|---|---|---|---|---|
| Age, median (IQR) | 39 (26–53) | 38.5 (26–52) | 39 (26–53) | 40 (25–51) | 0.97* |
| Male Gender | 206 (68.9) | 69 (70.4) | 90 (65.2) | 47 (74.6) | 0.38† |
| Race/Ethnicity | 0.25† | ||||
| White | 235 (78.6) | 80 (81.6) | 102 (74) | 53 (84.1) | |
| American Indian/Alaskan Native | 9 (3) | 3 (3.1) | 6 (4.3) | 0 | |
| Asian | 19 (6.4) | 3 (3.1) | 13 (9.4) | 3 (4.8) | |
| Black/African American | 11 (3.7) | 5 (5.1) | 6 (4.3) | 0 | |
| Native Hawaiian/Pacific Islander | 1 (0.3) | 0 | 1 (0.7) | 0 | |
| Hispanic | 24 (8) | 7 (7.1) | 10 (7.3) | 7 (11.1) | |
| % TBSA, median (IQR) | 7.1 (3–18) | 6.3 (3–16) | 7 (2.2–18.6) | 8 (3–17) | 0.67* |
P-value derived from ANOVA
P-value derived from χ2 test
Table 6.
Scar Outcome Measures by p27kip1 Genotype
| Total (N=300) |
CC (n=98, 33%) |
CA (n=139, 46%) |
AA (n=63, 21%) |
P-value* | |
|---|---|---|---|---|---|
| Worst Follow-up score on VSS, mean ± standard deviation | 7 ± 2.2 | 6.7 ± 2.4 | 7.1 ± 2.2 | 7.1 ± 2.1 | 0.31 |
| Worst Follow-up Vascularity Score, mean ± standard deviation | 2.1 ± 0.8 | 2 ± 0.9 | 2.1 ± 0.8 | 2.1 ± 0.8 | 0.67 |
| Worst Follow-up Pliability Score, mean ± standard deviation | 2.2 ± 1.1 | 2 ± 1.1 | 2.2 ± 1.1 | 2.2 ± 1.1 | 0.33 |
| Worst Follow-up Height Score, mean ± standard deviation | 1.1 ± 0.7 | 1 ± 0.7 | 1.1 ± 0.7 | 1 ± 0.6 | 0.68 |
| Worst Follow-up Itch Score, mean ± standard deviation | 3.7 ± 2.7 | 3.6 ± 2.8 | 3.8 ± 2.7 | 3.5 ± 2.7 | 0.77 |
P-value derived from ANOVA
Risk Factors for Hypertrophic Scar Development
Using multivariate logistic regression, we built a model for hypertrophic scar development as defined by a VSS score of greater than 7. Factors used in the model included age, gender, race/ethnicity, facial burn, percent TBSA burn ≥20, and p27kip1 genotype (Table 7). American Indian/Alaskan Native race had an odds ratio of 11.97 (95% Confidence Interval (CI); 1.42–100.82, P-value; 0.02) compared to Caucasian race. Facial burns carried an odds ratio of 9.67 (95% CI; 1.12–83.56, P-value; 0.039) compared to burns on the thorax or extremities. Subjects with greater than or equal to 20% TBSA burns had an odds ratio for hypertrophic scar development of 1.9 (95% CI; 1.01–3.57, P-value; 0.047) compared to those with less than 20% TBSA burns. P27kip1 genotype (either variant carriage or variant homozygosity) was not associated with a decreased risk of hypertrophic scar development. Additionally, age, gender, or any of the other race/ethnicity categories were not associated with hypertrophic scar development.
Table 7.
Risk of Hypertrophic Scar*
| Clinical Variable | Odds Ratio |
95% CI |
P value |
|---|---|---|---|
| Age | 0.97 | 0.76–1.24 | 0.82 |
| Male Gender | 1.17 | 0.66–2.12 | 0.58 |
| Race/Ethnicity (vs. Caucasian) | |||
| American Indian/Alaskan Native | 11.97 | 1.42–100.82 | 0.02 |
| Asian | 2.18 | 0.75–6.39 | 0.15 |
| Black/African American | 3.28 | 0.91–11.84 | 0.07 |
| Native Hawaiian/Pacific Islander | 1 | N/A | N/A |
| Hispanic | 0.61 | 0.23–1.65 | 0.33 |
| Facial Burn | 9.67 | 1.12–83.56 | 0.039 |
| >20% TBSA Burn | 1.9 | 1.01–3.57 | 0.047 |
| P27kip1 Genotype (vs. CC genotype) | |||
| Variant Carrier (CA) | 1.07 | 0.6–1.95 | 0.8 |
| Variant Homozygote (AA) | 1.07 | 0.52–2.2 | 0.86 |
By multivariate logistic regression analysis for hypertrophic scar defined as a Vancouver Scar Scale score >7
For our secondary analysis, we built a regression model for clinically significant itch using an itch score of >4. We included the same factors as in the regression for hypertrophic scarring (age, gender, race/ethnicity, facial burn, percent TBSA burn ≥20, and p27kip1 genotype; Table 8). The only factor associated with clinically significant itching was burn size greater than or equal to 20% TBSA (Odds ratio; 2.04, 95% CI; 1.09–3.81, P-value; 0.025).
Table 8.
Risk of Itch*
| Clinical Variable | Odds Ratio |
95% CI |
P value |
|---|---|---|---|
| Age | 0.86 | 0.68–1.09 | 0.23 |
| Male Gender | 0.93 | 0.53–1.62 | 0.8 |
| Race/Ethnicity (vs. Caucasian) | |||
| American Indian/Alaskan Native | 1.23 | 0.29–5.14 | 0.78 |
| Asian | 2.3 | 0.77–6.84 | 0.13 |
| Black/African American | 1.73 | 0.5–6.03 | 0.39 |
| Native Hawaiian/Pacific Islander | 1 | N/A | N/A |
| Hispanic | 1.7 | 0.7–4.17 | 0.24 |
| Facial Burn | 7.81 | 0.92–66.5 | 0.06 |
| >20% TBSA Burn | 2.04 | 1.09–3.81 | 0.025 |
| P27kip1 Genotype (vs. CC genotype) | |||
| Variant Carrier (CA) | 0.86 | 0.49–1.53 | 0.62 |
| Variant Homozygote (AA) | 0.86 | 0.43–1.73 | 0.68 |
By multivariate logistic regression analysis for clinically significant itch defined as an itch score of >4
DISCUSSION
No definitive treatment or sufficiently efficacious prevention strategy for hypertrophic scars exists. Whereas hypertrophic scars continue to present a significant problem to burn patients and their caregivers, advancing knowledge of clinical risk factors may prompt changes in practice patterns leading to more aggressive interventions in higher risk individuals. Further investigation into associated genetic variants could lead to targets for therapeutic interventions to prevent hypertrophic scar development.
Presence of p27kip1-838C>A was not associated with a decreased incidence of hypertrophic scar development in any genetic model. However, we have identified three novel independent risk factors for hypertrophic scarring; American Indian/Alaskan Native race, facial burns and burn size ≥20% TBSA.
P27kip1 is a cyclin-dependent kinase inhibitor that controls cell cycle progression. Degradation of the p27kip1 protein is required for a cell to transition from a quiescent to a proliferative state. The p27kip1 gene is located on chromosome 12. The p27kip1 SNP we were interested in, p27kip1-838C>A is located in the promoter region of the gene. Previous work has shown that substitution of the C to the A allele leads to increased potential transcription factor binding sites. Whereas the p27kip1 SNP has been associated with decreased risk of vascular fibroproliferation, our results suggest that this effect cannot be generalized to cutaneous scar formation. It is likely that the p27kip1 gene activity in smooth muscle cells involved in vascular fibrosis differs from cutaneous myofibroblasts involved in cutaneous wound healing. However, given that myofibroblast activity is similar to smooth muscle cells, this may warrant further mechanistic examination.
American Indian/Alaskan Native race has not been previously associated with increased risk of hypertrophic scar development. Likely, the number of American Indian/Alaskan Native patients sustaining burn injuries and treated in burn centers is too small for observation of significant associations; only 9 (3%) subjects in the current study identified themselves as American Indian or Alaskan Native. Our novel observation needs further investigation both in a validation study with a larger cohort and in a genome-wide association study to evaluate for potential genetic variants leading to this observation.
The association of facial burns with increased risk of hypertrophic scar development is also a novel finding. Potential explanations for this finding include site-specific differences in skin structure and function. Rinn and colleagues identified site-specific differences in the gene expression patterns of human fibroblasts. 14 Contraction has long been thought to contribute to HTS development and the face, especially the T-zone, is subjected to significant post-burn contractile forces. Another potential explanation for this finding includes differences in compliance of pressure garment therapy (PGT) between the face and other parts of the body leading to more appropriate treatment of HTS in other parts of the body. Johnson and colleagues reported a 41% compliance with PGT overall.15 More recent investigations into reasons for low compliance with therapy include embarrassment if the garment is visible, which is applicable to facial PCT.16,17 A prospective study would be valuable to control for pressure garment use and compliance.
We also identified an association between hypertrophic scar development and burn size, specifically burns over 20% TBSA, leading to a nearly 2-fold increased risk of HTS development. In addition, having ≥20% TBSA burns was associated with a 2-fold increased risk of clinically significant itch. Larger burn size has been has been associated with increased inflammatory reaction leading to increased morbidity and mortality.18 Similarly, exaggerated inflammatory responses to healing have been found in hypertrophic scars.19 These two interrelated observations serve as a likely explanation for this association.
Several potential shortcomings in this analysis must be identified. First, the relatively small cohort of 300 subjects could lead to missed associations, especially in the subgroup analysis. Whereas our post-hoc analysis showed that we were powered to identify differences in VSS and itch scores for the entire population, we were not powered to detect such differences in the subgroup analysis. Secondly, no Native American/Alaskan Native, Black/African American or Native Hawaiian/Pacific Islander subjects were homozygous for the variant allele in question. However, this is only a limitation if those patients exist and currently the NCBI database only lists European samples.20 Some studies advocate for evaluation of each racial group separately to avoid confounding due to racial differences in gene frequencies. We did perform a sub-analysis of the Caucasian subjects alone for associations of the p27kip1 SNP and HTS and did not see any associations in any genetic model (data not shown). Lastly, we chose to use a VSS score of greater than 7 as a way of identifying hypertrophic scar. This was based both on the distribution of scores in this cohort as well as clinical experience. However, there is no consensus in the literature as to what VSS score constitutes hypertrophic scarring.
In conclusion, Native American/Alaskan Native race, facial burns, and ≥20% TBSA burns were associated with increased risk of hypertrophic scar development. Knowledge of these novel risk factors for hypertrophic scar development may affect clinical decision making and operative management of higher risk patients. Furthermore, the association with a specific race should lead us to question whether there are yet-to-be-identified genetic variants at play.
Acknowledgments
This work was done with the financial support of the following: NIH (R01GM089704) & Washington State Council of Firefighters Burn Foundation
Footnotes
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The authors have no conflict of interests to declare.
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