| 1. Background and ObjectiveKeloid is a specific pathological scar in wound healing for human beingresulting from skin trauma. Keloid is much different from any other kind of scar tissue, it frequently persists at the site of injury, don't regress all the time; often recurs after excision, and always overgrows the boundaries of the original wound manifested by invasion of clinically normal skin. Keloid not only could results in the malformation and dysfunction which impact beautiful outlook, but also usually occurs pruritus and ache symptoms, which cause much physical and psychological anguish on patients with keloids. Therefore, keloid has been regarded as one of tricky problem to plastic surgery so far. Due to the inclarity of the causes for keloid formation, there is still no specific diagnostic method and valid therapeutic measure in clinic up to now. It is thus clear that to identify etiological factors for keloid will be the only way to diagnose exactly and treat effectually it.Many domestic and foreign scholars had carried out a series of investigations on the etiopathogenisis of keloid from a lot of aspects including in histology, pathology, physiology, immunology and genetics, et al., proposing a number of theories such as collagen synthesis and dissolution imbalance, cell growth factor, fibroblast defective apoptosis, immunity reaction and genetics, et al. However, all these theories only illustrated the respective relationship between keloid and etiopathogenisis, they could not demonstrate well the real causes for keloid formation. Most keloids occur sporadically, but some keloid cases are familial. Cosman had proved that the incidence rate of familial keloids was around 3% and higher occurrence in darker-skinned races. The characteristics on clinical genetics for keloids have only rarely been documented, and the mode of inheritance is not well known yet. Omo-Dare proposed an autosomal recessive inheritance pattern based on a collection of small pedigrees, whereas Bloom suggested an autosomal dominant inheritance pattern, based on an Italian family whose pedigree spanned 5 generations, the only reported large pedigree of a family with keloids at that time. Marneros found that the pattern of inheritance observed in 14 keloid pedigrees was consistent with an autosomal dominant mode with incomplete clinical penetrance and variable expression, the age of onset for keloids varied from early childhood to late adulthood, and most of the keloids presented at chest and shoulder areas. All these findings indicate that heredity is the major factor resulting in keloid formation, and the prevalence and expression for familial keloids are significantly different among various races. Thus, it will become a principal trend for us to ascertain the clinical genetic characteristics and locate the predispoing genes, screen and clone pathogenic gene in future study on etiopathogenisis of keloid.Recently, having conducted a genome-wide linkage screen to locate locus for genes predisposing to keloid formation in a Japanese and an African-American keloid family with microsatellite markers, Marneros identified linkage to chromosome 2q23 for the Japanese family and linkage to chromosome 7p11 for the African-American family, and presumed that one gene on chromosome 2q23 at 153cM (152Mbp) encoding for the TNF-a inhibitory protein 6 (TNFAIP6) might be a candidate gene for the Japanese keloid family, and another gene on chromosome 7p11 at 76cM (55Mbp) encoding for EGF receptor might be a candidate gene for the African-American keloid family. Exons, intron/exon junctions, and the promoter region of these two candidate genes were sequenced, and no mutations or disease-associated polymorphisms were identified however. Therefore, it was likely that mutations in other genes in the identified loci predispose to keloid formation in these two families. In addition, linkage to chromosome 7p11 and 2q23 was excluded in a moderately sized African-American keloid pedigree with ten affected family members. These results suggested that at least a third predisposing gene loci for keloid exists, demonstrating locus heterogeneity in familial keloid formation, in conformity to the clinical observation that the extent of keloid scarring was variable in different family. This study provides the first genetic evidence for keloid susceptibility loci and serves as a basis for the identification and location of such predisposing genes. On account of the significant differences among various races in keloid formation, whether the clinical genetic characteristics and locus for predisposing genes in Chinese keloid pedigrees are similar to what Marneros had detected in those keloid families remains unknown. Therefore, we performed an investigation on these aspects in six large Han Chinese keloid pedigrees. The affected member and generation in these families are more than those in previous genetic study on familial keloids, so they possess more representation and correspond better to the request on pedigree for genetic study about familial keloids in Chinese population. We identified that the pattern of inheritance observed in these Han Chinese keloid pedigrees was consistent with an autosomal dominant mode with incomplete clinical penetrance and variable expression. The identification for clinical phenotype and inherited pattern is the good groundwork for us to locate the predisposing genes of keloid. According to the research project on familal keloids, another doctor first selected two pedigrees (one with 5 affected generations and another with 4 affected generations) as subjects to perform a linkage analysis for keloid susceptibility locus with microsatellite markers on chromosome 2q23, and excluded linkage of these two pedigrees to chromosome 2q23. Therefore, we selected 32 members with more inherited significance from that pedigree containing in 5 affected generations as subjects to conduct a linkage analysis for keloid susceptibility locus by 4 microsatellite markers on and around chromosome 7p11 with the known maximal two-point LOD scores, and excluded linkage of the family to chromosome 7p11. Accordingly, we plan to identify and locate the locus for predispoing genes of keloid by analysising the linkages to the chromosome domains with the genes that have been considered may be closely associated with keloid formation.Owing to specific growth featere and clinical expression of keloid, it can be regarded as a benign tumor resulting from wound healing. Now, fibroblast has been proved to be the functional cell which contributes to the keloid formation. The unbalance of proliferation and apoptosis in fibroblast is the cytological substructure that leads keloid to unceasing accrementition and makes it hard to regress. Fas gene is a member of the tumor necrosis factor or nerve growth factor family receptor. The fibroblast will be induced to apoptosis after Fas receptor combines with its homologus ligand and monoclonal antibody. Apoptosis mediated by Fas gene has been considered to play an important role in fibroblast apoptosis. Fas gene is a single copy gene with 36 kilobases on chromosome 10q24.1. We identified and located predisposing genes for keloid by candidate gene cloning approach: Fas gene was assumed as a candidate gene resulting in predispotion to keloids, and four microsatellites next to the known genes (i. e. Fas, PTEN, BMPR1A and DNTT gene) relating with apoptosis defect or tumor formation in the 10Mbp region around Fas gene on chromosome 10q24.1 were selected as genetic markers. We wish to implement the destination to identify and locate the locus for predisposing genes of keloid by judging the linkage of the keloid pedigree to these markers on chromosome 10q24.1 with linkage analysis in order to supply with theoretical evidence for further correlated study about the virulence gene of keloid.2. Materials and Methods2.1 SubjectsThese six Chinese keloid pedigrees for studying clinical genetics are all from Han people among which there is no relationship. All the subjects had no recorded marriage history to other races of Chinese except Han people. The provincialism of these families consists of Liaoning (n=2), Neimenggu (n=1), Hebei (n=1), Fujian (n=1) and Heilongjian (n=1). Syndromes associated with keloids, namely Rubinstein-Taybi and Goeminne syndrome were not found in these families. Three pedigrees span 4 generations; two span 3 generations; and one, 5 generations. One pedigree displays keloids in 5 generations; two display in 4 generations; two display in 2 generations; and one, in 3 generations. These pedigrees account for 185 family members, 94 are male and 91 are female, of whom 45 display keloids and 5 don't present keloid as obligate unaffected carriers. Of the affected family members, 18 are male and 27 are female. The pedigree for locating predispoing genes of keloid comes from Neimenggu. This family displays keloids in 5 generations, accounts for 63 family members, 33 are male and 30 are female, of whom 14 display keloids and 3 don't present keloid as obligate unaffected carriers, and of the affected family members, 7 are male and 7 are female. 32 members with more inherited significance in this family were selected as subjects to locate the predisposing genes of keloid. This study was approved by all institutional review boards. All family members who were interviewed and examined signed an informed consent to participate in the study.2.2 Methods2.2.1 Ascertainment for clinical phenotype of keloid and collection for keloid pedigreesFor this study, pedigree members were classified as keloid former (affected) or nonkeloid former (unaffected). The affected and unaffected family members were carefully examined and precisely diagnosed by 2 plastic surgeons with substantial clinical experience treating keloids. The diagnosis was made clinically, based on the criterion that the scar extended beyond the boundaries of the original injury manifested by invasion of clinically normal skin or would not regress spontaneously after a year, or recurred after excision. Sites of keloid formation were documented by photography. Detailed inquiry was made into the family history, and information was collected about deceased or unavailable individuals. Individual clinical information was kept in a file. Peripheral venous blood samples from 32 members with more inherited significance in that family with 5 affected generations were collected for further study of keloid on molecular genetics. In some cases, histological specimens from keloids of affected family members were obtained in order to confirm histologically. The use of human tissue/subjects adhered to the Declaration of Helsiniki Guidelines in correlated researches.2.2.2 Construction of pedigree charts and analysis for the mode of inheritanceTo analyze the mode of inheritance, pedigree charts were constructed from the obtained datum. Unaffected children and adolescents were included in the pedigrees, but were not considered in the analysis of the inheritance pattern because they might clinically express keloids as they age. Unaffected family members with an affected parent and at least 1 affected child were regarded as obligate carriers.2.2.3 Extraction of genomic DNA Genomic DNA was extracted by Classic Phol/Chl Law with 2 ml peripheral venous blood sample from every subject (EDTA anticoagulation).2.2.4 Selections of microsatellite markers and synthese for primersReferring to recent pertinent literatures and bioinformatics databases, 4 microsatellite markers within the domain of 18.8Mbp on and around chromosome 7p11 with the known maximal two-point LOD scores and 4 microsatellite markers next to the known genes relating with apoptosis defect or tumor formation within the region of 10Mbp around Fas gene on chromosomes 10q24.1 were selected as polymorphic genetic markers to locate the locus of predisposing genes for keloid. The forward and reverse sequencesfor the primers were obtained also, and the homologous primers were synthesesed then.2.2.5 PCR amplifications, detections of amplified products and genotypingsThese markers were amplified by PCR in PE9700. The amplified products were processed by Genescan (Ver.3.11) and Genotyper (Ver.3.7) for getting the size of each fragment and genetype.2.2.6 Data processingThe eligible genotyping datum were used to calculate the two-point LOD score for every marker by the MLINK option of the LINKAGE package (Ver.5.11) with an autosomal dominant mode, a phenocopy rate of 1%, a disease allete frequency of 0.1%, and a penetrance rate of 90%, atθ=0.000～0.400, in accordance with the recent similar study by Marneros. Linkages of the keloid pedigree to these markers were judged by respective LOD score.3. Results3.1 Genetic characteristicsThere is no statistical difference between male and female in prevalence of keloid. The affected family member can be heterozygous, and a child of an affected and unaffected parent has a 50% chance of being affected. One of parents with affected children also presented keloids in most families, whereas one of parents with affected children mostly displayed keloids in minor families, and the others who didn't present keloids were obligate unaffected gene carriers (n=5). The trait of keloid was transmitted continuously in three pedigrees, transmitted interruptedly in two pedigrees, and has been transmitted continuously in three generations in one pedigree with 4 generations.3.2 Age of first onsetOf the 45 affected family members, 37 (82.22%) cases first presented keloids between 16 and 25 years of age, which was just the adolescence for Chinese population.3.3 Inducements of first onsetOf the 45 affected family members, 38 (84.44%) cases were induced by acne and folliculitis.3.4 Sites of displayOf the 45 affected family members, 21 (46.67%) cases displayed keloids at prothorax; 7 (15.56%) at shoulder and back; 5 (11.11%) at face and neck; 5 (11.11%) at limbs; 3 (6.67%) at prothorax and back; 2 (4.44%) at face, neck, chest, back, abdomen and limbs; 1 (2.22%) at face, neck and shoulder; and 1 (2.22%) at abdomen. 3.5 Variable clinical expressionThe clinical severity of keloid formation differs between these families, as well as within a family. The clinical expression of the proband in each keloid pedigree was most typical and severe than any other affected member in the same family. Furthermore, the keloids in the same pedigree presented the reduced clinical phenotypes one generation after another generation.3.6. Morphologic featuresMost of chest keloids were dumbbell-shaped, back scars butterfly-shaped, shoulder scars propeller-shaped, face and neck scars trabs-shaped, limbs scars propeller-shaped, and abdomen scars irregular in these familial keloids.3.7 Tendency of symmetrical invasionOf the 45 affected family members, 29 (66.44%) presented the tendency which the keloids proximally occurred symmetrically around the anterior and posterior central axis of body.3.8 Linkage analysesAtθ=0.000～0.100, the maximal two-point LOD scores for microsatellite markers D7S181, D7S499, D7S494 and D7S3046 were all less than -2, which excluded linkage of the keloid pedigree to these markers; for D10S1765 and D10S1735 were 1.743 and 1.218 respectively, which supported linkage of the keloid pedigree to the region about 1Mbp between D10S1765 and D10S1735; for D10S1562 was 0.391, which may excluded linkage of the keloid pedigree to this marker; and for D10S1687 was less than -2, which excluded linkage of the keloid pedigree to this marker.4. Conclusion4.1 The pattern of inheritance observed in these Han Chinese keloid pedigrees is consistent with an autosomal dominant mode with incomplete clinical penetrance and variable expression. Furthermore, the keloids in the same pedigree presented the reduced clinical phenotypes one generation after another generation.4.2 The keloids for these pedigrees mainly occur spontaneously during adolescence between 16 and 25 years of age, predispose to display at prothorax, back and shoulder with respective specific figures, and often present the tendency of proximally symmetrical invasion.4.3 The possibility on chromosome 7p11 for the predisposing genes in these Han Chinese keloid pedigrees may be excluded.4.4 The predisposing genes for these Han Chinese keloid pedigrees may be mapped to the region about 1Mbp on chromosome 10q24.1 between markers D10S1765 and D10S1735, suggesting the PTEN gene and Fas gene may be the candidate genes for these pedigrees.4.5 These findings demonstrate genetic heterogeneity exists in keloid formation.
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