Keloid formation [MIM 148100] is a common scarring disorder with high incidence in African-Americans and Asians. Keloids often occur sporadically, but a genetic predisposition for keloid formation has been considered, mainly because of higher incidence in darker-skinned populations (reviewed byKelly, 1988). Keloids usually form after trauma, and are considered to be a result of altered wound healing with excessive scar tissue formation that extends beyond the area of the initial wound and does not regress spontaneously (Cosman et al, 1961;Peacock et al, 1970). Some keloids can involve large areas of the body, despite a minor wound site, and have a high rate of recurrence after surgical excision (English and Shenefelt, 1999). In contrast, hypertrophic scars do not extend beyond the original wound area, commonly regress spontaneously, and rarely recur after excision.
In this study, we distinguished hypertrophic scars from keloids clinically, and studied only families with keloids. The formation of keloids is influenced by many different factors, such as age, ethnicity, anatomic location, and type of trauma. The pathogenetic mechanisms that lead to keloid formation are still unknown however.
When compared to normal dermal fibroblasts or to fibroblasts derived from hypertrophic scars, keloid fibroblasts showed in vitro many alterations in growth factor response (Russell et al, 1988;Babu et al, 1992;Haisa et al, 1994;Kikuchi et al, 1995), cytokine production (Lee et al, 1999), extracellular matrix production (Uitto et al, 1985;Babu et al, 1989;Bettinger et al, 1996), proliferation (Bettinger et al, 1996), and apoptosis (Chodon et al, 2000;Ishihara et al, 2000). It remains unknown as to why keloid fibroblasts show such differences compared to normal dermal fibroblasts or hypertrophic scar fibroblasts.
In order to identify genes that are involved in the pathogenetic mechanisms of keloid formation and predispose to this scarring disorder, we have chosen a genetic approach and studied families with a high occurrence of keloids. In the families studied we found that keloid formation occurred with an autosomal dominant mode of inheritance (Marneros et al, 2001). The inheritance mode of keloid formation in these families suggested that a genetic linkage approach could be used to identify keloid susceptibility loci, and that mutations in single genes can predispose to this scarring disorder.
No gene locus for familial keloid formation has been reported so far. Thus, we performed a genome-wide linkage search for genes predisposing to keloid formation in two large families, and identified keloid susceptibility loci on chromosomes 2q23 and 7p11.
Results
Clinical findings
The clinical severity of keloid formation differed in the families identified. In the Japanese family with keloids (family A), all seven affected family members had only small to moderately sized keloids Figure 1a and full penetrance could be observed. Their keloids resulted from minor trauma, vaccination, or minor surgery. One keloid has remained for 30 y. In contrast, in the large African-American family (family B) we found a high variation in severity of keloid formation, with some family members having only small earlobe keloids, whereas other family members had very large keloids that involved extended areas of the body at several sites Figure 1b. Family B accounts for 13 affected members and two obligate carriers. Most of the affected members in this family presented with multiple large keloids, which are particularly large in three family members. For some affected individuals, minor trauma (e.g., chickenpox vaccination) led to keloid formation, whereas for others only major wounds (e.g., abdominal hysterectomy surgery) resulted in keloids. There was no relationship between these two families. Based on the difference in clinical presentation of keloids between families, and the variable incidence of keloids in different populations, we speculated that alterations in more than one gene might result in a predisposition for keloid formation.
Figure 1.
Keloids in a member of family A and in a member of family B. (A) Chest keloid of moderate size in family member #10 of the Japanese family A. (B) Extensive keloids on the trunk, the neck, and the arms of family member #13 of the African-American family B.
Full figure and legend (193K)Linkage of the Japanese keloid family to chromosome 2q23
To identify loci for genes predisposing to keloids and to test for possible locus heterogeneity, we conducted a genome-wide linkage screen in these two families. The initial genome-wide linkage analysis of the Japanese family A resulted in two-point LOD scores >2 for a single chromosomal region on 2q23. Four markers on chromosome 2q23 resulted in LOD scores >1, within an interval of 32 cM between D2S1328 (133 cM; 126 Mbp) and D2S1353 (165 cM; 160 Mbp). Marker D2S1399 (152 cM; 148 Mbp) resulted in a maximal two-point LOD score of 3.01 at 
=0 (penetrance 100%; phenocopy rate 0%), which is also the calculated theoretical maximal two-point LOD score for this pedigree. Assuming a phenocopy rate of 1%, the two-point LOD score for this marker was 2.99 (=0), and with 95% penetrance and 1% phenocopy rate, the two-point LOD score was 2.86 (=0). Other genomic regions that resulted in positive two-point LOD scores between 1 and 2 were investigated by genotyping additional microsatellite markers in these regions (chromosome 13 at 99 cM with a two-point LOD score of 1.59 (=0); chromosome 14 at 56 cM with a two-point LOD score of 1.23 (=0)). Haplotype analysis excluded linkage for these regions. Thus, the genome-wide screen for family A resulted in a single candidate region on chromosome 2q23. To evaluate this region further, eight additional microsatellite markers in this locus were analyzed. The order and the distance between the markers were obtained from the Marshfield Genetic Database and the CSC Genome Database, and they are, in order from centromeric to telomeric, D2S1394 (91 cM; 73 Mbp), D2S1790 (103 cM; 85 Mbp), D2S2972 (114 cM; 102 Mbp), D2S410 (125 cM; 116 Mbp), D2S349 (150 cM; 142 Mbp), D2S132 (151 cM; 145 Mbp), D2S356 (153 cM; 152 Mbp), and D2S2275 (154 cM; 152 Mbp). Meiotic recombinants place the gene predisposing to keloids in this family within a 40 cM interval bounded by D2S410 and D2S1353, and refine the minimal interval encompassing the keloid predisposition gene to the region between D2S1328 and D2S2275. Haplotype analysis showed a consistent disease allele for all affected family members between D2S410 and D2S1353 Figure 2. Assuming 95% penetrance and 1% phenocopy rate, two-point LOD scores for these markers were calculated Table I. Centromeric to D2S1399, marker D2S349 resulted in a two-point LOD score of 2.84 (=0), and telomeric to this region marker D2S356 resulted in a two-point LOD score of 2.84 (=0). Multipoint linkage analysis with these three markers resulted in a maximal multipoint LOD score of 2.86 at 152 cM. We analyzed the genomic region between the meiotic recombinants and close to the marker D2S1399, which yielded the maximal two-point LOD score in this pedigree, for genes that may be candidates for a predisposition to keloid formation. A number of candidate genes that could have a role in wound healing were found in this locus. One candidate gene in this locus encodes for the TNF-
inhibitory protein 6, TNFAIP6, and is located on chromosome 2q23 at 153 cM (152 Mbp). This gene with a potential role in wound healing has a hyaluronan-binding domain, which is known to be involved in extracellular matrix stability and cell migration, and the expression of this gene can be induced by TNF- and IL-1 (Lee et al, 1993).
Figure 2.
Genotypes of family A for markers on chromosome 2q23. Genotypes for 11 markers from chromosome 2q23 in the Japanese keloid family A. Blackened symbols indicate individuals affected with keloids. Vertical bars next to the alleles indicate the segregating haplotypes between affected individuals. Critical meiotic recombinants in family members #40 and #60 refine the minimal interval encompassing the keloid predisposition gene to the region between D2S1328 and D2S2275.
Full figure and legend (37K)
Linkage of the African-American keloid family to chromosome 7p11
Haplotype analysis excluded linkage of the African-American family (family B) to the locus on chromosome 2q23. Thus, we searched for another linked locus in family B. Genome-wide linkage analysis identified only a single chromosomal region with two-point LOD scores >2. On chromosome 7p11, marker D7S3046 (79 cM; 68 Mbp) revealed a two-point LOD score of 2.72 at =0 (penetrance 90%, phenocopy rate 3%). The flanking centromeric marker was D7S2204 (91 cM; 78 Mbp) and resulted in a two-point LOD score of 1.59 (=0). The flanking telomeric marker was D7S1818 (70 cM; 49 Mbp) and resulted in a two-point LOD score of 1.77 (=0). The maximal multipoint LOD score with these three markers was 2.72 between 79.0 and 81.4 cM. Haplotype analysis and extensive genotyping with additional microsatellite markers in other loci identified in this screen with positive LOD scores between 1 and 2 failed to detect any linkage (chromosome 18 at 75 cM with a two-point LOD score of 1.86 (=0.1)). Thus, chromosome 7p11 is the major keloid susceptibility locus for this family.
To evaluate these initial linkage results further, 15 additional microsatellite markers in this region were analyzed. The order and the distance between these markers were obtained from the Marshfield Genetic Database and the CSC Genome Database, and they are, in order from telomeric to centromeric, D7S691 (64 cM; 42 Mbp), D7S678 (61 cM; 43 Mbp), D7S1793 (70 cM; not mapped in CSCGD), D7S1830 (73 cM; 51 Mbp), D7S506 (74 cM; 52 Mbp), D7S2475 (74 cM; 53 Mbp), D7S2552 (74 cM; 54 Mbp), D7S2550 (75 cM; 54 Mbp), D7S499 (76 cM; 55 Mbp), D7S659 (76 cM; 55 Mbp), D7S473 (77 cM; 57 Mbp), D7S494 (77 cM; 57 Mbp), D7S2429 (77 cM; 62 Mbp), D7S2530 (78 cM; 63 Mbp), and D7S820 (98 cM; 83 Mbp). Marker D7S499 resulted in a maximal two-point LOD score of 3.16 (=0). Marker D7S494 resulted in a two-point LOD score of 2.38 (=0), and marker D7S2475 resulted in a two-point LOD score of 1.60 (=0) Table II. A maximal multipoint LOD score of 3.16 was found for the interval between D7S499 and D7S494. Haplotype analysis was performed based on genotypes of all tested markers on 7p11. Meiotic recombinants place the gene predisposing to keloids in this family within a 16 cM interval between D7S678 and D7S494, and refine the minimal interval encompassing the keloid predisposition gene to the region between D7S1818 and D7S473. Haplotype analysis showed a consistent disease allele for all affected family members in this genomic interval, with the exception of one affected family member (#1002) Figure 3. This family member had developed a single keloid at age 30 (and was thus handled as an affected family member in the linkage calculations), but does not share the disease-linked allele with affected individuals in this locus. Since all the other 19 family members showed consistent haplotypes in this region, we suggest that this family member represents a phenocopy. The keloid in this family member might have resulted from a sporadic mutation or be inherited through the unknown father. Epidemiological studies have shown that keloid formation has a frequency of 3%–6% in the African-American population (reviewed byKelly, 1988). Due to this high occurrence of keloids in the African-American population, it is likely that this individual represents a phenocopy, particularly because of the lack of any other linked genomic region in this pedigree and because of the positive two-point LOD scores at chromosome 7p11. The marker with the peak two-point LOD score was D7S499 at 76 cM. A strong candidate gene, the EGF receptor gene (76 cM; 55 Mbp), is linked to this marker. Several studies provide evidence for a role of the EGF signaling pathway in keloid pathogenesis. Cell culture studies demonstrated that EGF induces a significantly greater growth response in keloid fibroblasts than in normal fibroblasts (Kikuchi et al, 1995).
Figure 3.
Genotypes of family B for markers on chromosome 7p11. Genotypes for 16 markers from chromosome 7p11 in the African-American keloid family B. Critical meiotic recombinants in family members #16 and #105 refine the minimal interval encompassing the keloid predisposition gene to the region between D7S1818 and D7S473. Family member #1002 does not share segregating haplotypes between affected family members, possibly representing a phenocopy.
Full figure and legend (73K)Discussion
Keloid formation is a common scarring disorder that can occur with an autosomal dominant inheritance pattern in some families (Marneros et al, 2001). The genes predisposing to keloid formation in such families are unknown. Weconducted a genome-wide linkage search for keloid susceptibility genes in two large unrelated families, which showed differences in the extent and penetrance of keloid formation.
The Japanese family A showed linkage to marker D2S1399 at chromosome 2q23 with a maximal two-point LOD score of 3.01 at =0. Haplotype analysis showed a consistent disease allele for all affected family members within a 40 cM interval between D2S410 and D2S1353. This locus contains genes that have been implicated to play a role in the regulation of wound healing responses and are therefore candidate genes for familial keloids. One such gene encodes for the TNF- inhibitory protein 6, TNFAIP6, which has been mapped closely to the marker on chromosome 2q23 that yielded the peak LOD score for the Japanese pedigree. This gene was originally identified as a tumor necrosis factor (TNF)-inducible gene in human fibroblasts (Lee et al, 1992), and human recombinant TNFAIP6 showed a potent anti-inflammatory activity in IL-1-induced acute inflammation (Wisniewski et al, 1996).
The African-American family B showed no linkage to chromosome 2q23. We identified linkage of this pedigree to chromosome 7p11. Marker D7S499 on chromosome 7p11 resulted in a maximal two-point LOD score of 3.16 at =0. This marker is linked to the gene for the EGF receptor. Interestingly, keloid fibroblasts show a higher proliferation rate in response to EGF than normal fibroblasts (Kikuchi et al, 1995). In addition, it has been reported that keloid fibroblasts express higher levels of EGF receptor compared to normal dermal fibroblasts (Chin et al, 2000). These reports suggest that the gene for the EGF receptor is a candidate gene for susceptibility to keloid formation.
Thus, we screened genomic DNA of affected and unaffected family members in the Japanese family for mutations in the TNFAIP6 gene, and family members in the African-American family for mutations in the EGFR gene. Exons, intron/exon junctions, and the promoter region of the genes were sequenced as described previously (Ueki et al, 2001). No mutations or disease-associated polymorphisms were identified however (not shown). Therefore, it is likely that mutations in other genes in the identified loci predispose to keloid formation in these two families.
We analyzed additional moderately sized African-American keloid pedigrees for linkage to the identified loci on chromosomes 7p11 and 2q23. Linkage analysis in a small African-American pedigree with six affected family members did not exclude linkage to chromosome 7p11 (two-point LOD score of 0.52 (=0) at 70 cM). Analysis of another small African-American pedigree with three affected family members yielded a two-point LOD score of 0.56 (=0) for a marker at 70 cM on chromosome 7p11 (not shown). Haplotype analysis in these two additional African-American pedigrees, however, did not narrow the critical interval for the locus on chromosome 7p11.
Based on the high occurrence of keloids in the African-American population and the observed variation in severity of keloid formation, it is likely that additional loci for keloids exist. In fact, we excluded linkage to chromosomes 7p11 and 2q23 in a moderately sized African-American pedigree with ten affected family members (not shown). These results suggest that at least a third keloid locus exists. Thus, the identification of additional keloid families is needed to further narrow the size of the identified loci on chromosomes 7p11 and 2q23, and to map further keloid susceptibility loci.
The genome scans conducted in these two families provide the first genetic evidence for keloid susceptibility loci. Our data demonstrate locus heterogeneity in familial keloid formation, consistent with the clinical observation that the extent of keloid scarring is variable in different families. Despite detailed histological and biochemical analyses of keloid tissue and keloid fibroblasts in culture, the causes for keloid formation remain unknown. It is likely that several factors influence the development and extent of the formation of a keloid in a predisposed individual, such as age or anatomic location of trauma. The identification of families with an autosomal dominant inheritance pattern of keloid formation suggests, however, that mutations in single genes can predispose to this scarring disorder. The characterization of the keloid susceptibility loci identified in this study provides the basis for the identification of such predisposing genes, which should significantly enhance our knowledge about the pathogenetic mechanisms involved in keloid formation. This may not only result in better treatment options for this common scarring disorder, but also provide new insights into the complex mechanisms of wound healing. Identifying these genes will lead to a better understanding of the biological mechanisms that regulate scarring.
Materials and Methods
Subjects
A Japanese family (family A) was identified through probands from Tokyo, Japan. An African-American family (family B) was identified through probands from New York City, USA. This study was approved by the appropriate institutional review boards, and informed consent was obtained before collecting blood samples from participating family members. For this study, affected and unaffected family members were carefully examined by dermatologists and plastic surgeons, who had substantial clinical experience in the diagnosis and treatment of keloids. The diagnosis was made clinically, based on the criterion that the scar extended beyond the boundaries of the original injury. In some family members it was possible to obtain a biopsy from a keloid and to confirm the diagnosis histologically, with collagen fibers appearing thicker and more irregular than in the normal dermis (Marneros et al, 2001). As keloids commonly occur between ages 10 and 30 y, unaffected children and adolescents were included in the pedigrees, but were considered uninformative in the genetic analysis because they might clinically express keloids at a later age. Unaffected family members with an affected parent and at least one affected child were regarded as obligate carriers in this study. After informed consent was obtained, we collected venous blood samples and extracted genomic DNA, as previously described (Tiziani et al, 1999). The use of human tissues/subjects adhered to the Declaration of Helsinki Guidelines.
Genotyping
For the initial genome-wide scan, microsatellite markers from the Marshfield Weber screening set 10, spaced at 10 cM intervals, were amplified by PCR, using standard protocols (Weber and Broman, 2001). To evaluate the initial linkage results further, additional microsatellite markers were analyzed in regions with suggestive linkage (LOD score >1). The order and the distance between these markers were obtained from the Marshfield Genetic Database and the CSC Genome Database.
Linkage analysis
Autosomal dominant inheritance was assumed for these pedigrees. Two-point LOD scores were calculated using the MLINK option of the LINKAGE package (Lathrop and Lalouel, 1984), and multipoint LOD scores were generated using the VITESSE program (O'Connell and Weeks, 1995). Two obligate carriers (unaffected individuals with affected parent and child) were identified in the African-American pedigree. Therefore, in the genome-wide linkage calculations for this family, keloid disease was modeled with penetrance rates between 70% and 95%. The estimated occurrence of keloids in the African-American population is less than 6% (Marneros et al, 2001). Thus, we varied phenocopy rates between 3% and 6%. The disease allele frequency was estimated to be 0.1%, with equal recombination fractions in male and female individuals. Two-point LOD score analysis was performed using calculated allele frequencies from a test population (174 individuals), or with equal allele frequencies. In genome-wide LOD score calculations for the Japanese family, keloid disease was modeled with different calculation parameters, because full penetrance was observed. Calculations were performed with penetrance rates of either 95% or 100%, and with phenocopy rates of either 0% or 1%.
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Appendices
Appendix: Electronic Database Information
URLs for data used in this study are as follows: Center for Medical Genetics, Marshfield Medical Research Foundation, http://research.marshfieldclinic.org/genetics/ (for identification and order of microsatellite markers); Ensembl, http://www.ensembl.org/ (for identification of candidate genes); Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/ (for Keloid [MIM 148100]); CSC Genome Bioinformatics, http://genome.cse.ucsc.edu/ (for identification of candidate genes).
Acknowledgments
We are grateful to all the family members for their cooperation in this research. We are thankful to the members of the Marshfield Genetics Center, and to Dr Steve de Palma for advice on linkage analysis. We are grateful to Dr Miikka Vikkula for a critical reading of the manuscript. We are especially thankful to Dr Thomas Krieg and Dr Beate Eckes for advice and continuous support. This work was supported by grants AR36819, AR36820, and AR45286 from the National Institutes of Health, Bethesda, MD; by a grant from the Koeln Fortune Program of the University of Cologne Medical School, Germany; and by the Boehringer Ingelheim Fonds.
