汉族瘢痕疙瘩家系临床遗传学及7p11和10q24.1易感基因定位研究
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摘要
一、研究背景及目的
瘢痕疙瘩是人类特有的一种创伤后病理性瘢痕愈合现象，它不同于其它瘢痕组织，具有过度生长、超过原伤口界限、并侵犯邻近正常皮肤组织、始终不退化和单纯手术切除后易复发等特点。瘢痕疙瘩不仅可继发畸形和功能障碍，影响美观，而且常伴有明显的瘙痒和疼痛症状，给患者造成极大的身心痛苦，是整形外科长期以来面临的重大难题之一。然而，因其病因至今仍不甚清楚，所以目前临床上尚缺乏特异性高的诊断方法和有效性强的治疗措施。可见，明确瘢痕疙瘩的病因将是准确诊断和有效治疗瘢痕疙瘩的根本途径。
关于瘢痕疙瘩的病因，国内外许多学者已经从组织学、病理学、生理学、免疫学和遗传学等方面开展了一系列的研究，提出了诸如胶原合成与降解失衡、细胞因子、成纤维细胞凋亡障碍、免疫反应和遗传学等许多学说，但这些学说都无法阐明瘢痕疙瘩发病的真正原因。瘢痕疙瘩大多散发发病，但具有一定的家族遗传倾向，Cosman等证实其家族性发生率约为3％，深肤色人种多发。有关瘢痕疙瘩的临床遗传学特征至今仍少有报道，而且其遗传模式仍未完全明了。Bloom通过对当时所报道的仅有一个五代瘢痕疙瘩意大利家系进行分析后认为它属于常染色体显性遗传，然而，Omo-Dare对所收集的瘢痕疙瘩小家系分析后认为它属于常染色体隐性遗传。Marneros等对14个瘢痕疙瘩家系进行临床遗传学方面的研究后发现，它们的遗传模式符合常染色体显性遗传伴外显不完全，且表现度存在差异，从幼童期到成年后期均可发病，大多数瘢痕疙瘩发生于胸部和肩部。由此可见，遗传是瘢痕疙瘩发病的主要因素，患病率和临床表型存在显著的种族差异。明确临床遗传学特征、定位易感基因、筛查和克隆致病基因等将成为今后瘢痕疙瘩病因研究的主要方向。
最近，Marneros等选择微卫星标记通过全基因组扫描和连锁分析对一个瘢痕疙瘩日本家系和一个非洲裔美国人家系进行易感基因的定位研究后发现，日本家系与染色体2q23连锁，非洲裔美国人家系与染色体7p11连锁，并认为位于染色体2q23上153cM(152Mbp)处的一个转录为肿瘤坏死因子-a抑制蛋白6(TNFAIP6)基因和位于染色体7p11上76cM(55Mbp)处的表皮生长因子(EGF)受体基因分别是日本家系和非洲裔美国人家系的一个候选基因，但是对这两个候选基因的外显子、内含子与外显子的拼接点及启动子序列进行测序，并未发现突变或与疾病相关的多态性存在，因此，认为可能在上述这两个候选基因位点附近其它基因的突变才导致对瘢痕疙瘩的易感。另外，他们还报道了对一个有10人发病的非洲裔美国人中等大小的瘢痕疙瘩家系进行同样的研究，却没发现该家系与染色体2q23和7p11存在连锁关系，提示至少有第三个瘢痕疙瘩易感基因位点存在，说明瘢痕疙瘩存在遗传异质性，这与临床上观察到不同家系瘢痕疙瘩病情不一的现象相一致。该研究首次提出了存在瘢痕疙瘩易感基因位点的遗传学证据，对进一步识别和定位瘢痕疙瘩的易感基因具有指导意义。由于瘢痕疙瘩的发病存在显著的种族差异，中国人群瘢痕疙瘩家系的临床遗传学特征和易感基因位点是否与Marneros等的研究发现相似，目前尚不清楚。为此，我们收集了六个中国汉族瘢痕疙瘩大家系进行这方面的研究，这些家系规模大、发病人数多，能较好地满足遗传病研究对家系的要求，对中国汉族人群瘢痕疙瘩家系的遗传学方面研究，具有较好的代表性。我们的研究发现，这些中国汉族人群瘢痕疙瘩家系的遗传模式也符合常染色体显性遗传伴外显不完全，且表现度存在差异。瘢痕疙瘩临床表型和遗传模式的确定为进一步开展其易感基因的定位研究奠定了良好的基础。
按照课题研究计划，由另一位博士研究生首先选择其中1个5代发病和1个4代发病的家系作为研究对象，也采用微卫星标记，通过连锁分析，否定了这两个中国汉族瘢痕疙瘩家系与染色体2q23的连锁关系。因此，本研究从上述那个5代发病家系中选择具有较高遗传学研究意义的32名成员作为研究对象，在Marneros等研究发现的基础上，于染色体7p11及其周围选取已知的四个最大两点LOD值的微卫星作为遗传标记，通过连锁分析，也否定了这个中国汉族瘢痕疙瘩家系与染色体7p11的连锁关系。为此，我们又从分析家系与目前被认为和瘢痕疙瘩发病可能存在密切关系的基因所在的染色体区域的连锁关系之途径来识别和定位瘢痕疙瘩的易感基因。
由于瘢痕疙瘩所具有的独特生长特性及临床表现，因此它可被视为一种创伤愈合后形成的良性肿瘤。现已证明成纤维细胞是瘢痕疙瘩形成的功能性细胞，其增殖-凋亡调控的失衡是导致瘢痕疙瘩不断增生而且难以退化的细胞学基础。Fas基因属于NGF／TNF受体家族，其相应的配体及单克隆抗体与之结合后可诱导细胞凋亡。Fas介导的凋亡被认为在成纤维细胞的凋亡中发挥着重要的作用。Fas基因位于染色体10q24.1上，是全长36kb的单拷贝基因。我们采用候选基因克隆策略来识别和定位瘢痕疙瘩易感基因，即假定Fas基因为导致瘢痕疙瘩易感的一个候选基因，选择位于10q24.1上Fas基因及其周围共约10Mbp的染色体区域内已知的与细胞凋亡障碍或肿瘤发生有关的基因(即Fas、PTEN、BMPR1A和DNTT基因)紧邻的4个微卫星作为遗传标记，通过连锁分析判断家系与这些微卫星位点之间是否存在连锁关系，以期识别和定位瘢痕疙瘩的易感基因，为进一步的致病基因相关研究提供理论依据。
二、材料与方法
1．研究对象
用于临床遗传学研究的六个中国人群瘢痕疙瘩家系中，来自辽宁2个、内蒙古、河北、福建和黑龙江各1个，均为汉族人群，家系之间无亲缘关系，家系成员无与汉族以外人种婚配史，无Rubinstein-Taybi和Goeminne综合症(伴发有瘢痕疙瘩)。在这六个家系中，5代家系1个，4代家系3个，3代家系2个；5代发病家系1个，4代发病家系2个，3代发病家系2个，2代发病家系1个；家系成员185个，男94个，女91个；发病45个，男18个，女27个，未发病的基因携带者5个。用于定位瘢痕疙瘩易感基因的家系来自内蒙古，5代发病，家系成员63个，男33个，女30个；发病14个，男7个，女7个；未发病的基因携带者3个；从该家系中选择具有较高遗传学研究意义的32名成员作为研究对象。本研究事先得到相关审核部门的同意，所有参与研究的家系成员均签署了知情同意书。
2．方法
(1)瘢痕疙瘩临床表型的确定和家系的收集
由2名对瘢痕疙瘩诊断和治疗具有丰富临床经验的整形外科医师，根据瘢痕疙瘩临床诊断标准(即瘢痕超过原损伤范围并向周围正常皮肤侵犯，或瘢痕病程超过1年仍无自发消退征象，或术后瘢痕复发者)把家系成员分为瘢痕疙瘩发病者和未发病者，对所有发病和未发病的家系成员进行详细的病史询问、全面的体格检查和准确的临床诊断，并对瘢痕疙瘩发病部位照相取证，建立个人临床信息资料档案(包括已故和无法到访的成员)。采集那个5代发病家系中具有较高遗传学研究意义的32名成员的外周静脉血供进一步的分子遗传学研究，并对部分患者的瘢痕疙瘩组织取活检行组织病理学检查以确诊，遵照赫尔辛基宣言准则使用这些人体组织进行相关的研究。
(2)绘制家系系谱图和分析遗传模式
根据收集的临床资料，绘制家系系谱以研究瘢痕疙瘩的遗传模式。虽然系谱包括未发病的儿童和青少年，但在分析遗传模式时予除外，因为随着年龄的增加，他们可能出现瘢痕疙瘩临床表型。未发病的家系成员其父母之一发病且至少有1个子女发病则称他或她为肯定携带者。
(3)提取基因组DNA
抽取每位家系成员的外周静脉血2ml(EDTA抗凝)，采用经典的酚氯仿法提取基因组DNA。
(4)选择微卫星标记和合成引物
通过查阅最近的相关文献和生物信息学数据库，在染色体7p11及其周围共18.8Mbp区域内选取4个已知的最大两点LOD值的微卫星和在Fas基因所在的染色体区带10q24.1及其周围共10Mbp的区域内选择4个与细胞凋亡障碍有关的已知基因相邻的微卫星作为多态遗传标记，用于定位瘢痕疙瘩易感基因研究；并查阅这些微卫星标记的上下游引物序列，以合成相应的引物。
(5)微卫星标记PCR扩增、产物检测和基因型分型
PCR扩增在PE9700型PCR反应仪中进行，产物经Genescan(Ver.3.11)和Genotyper(Ver.3.7)软件处理后根据检测片段大小进行基因型分型。
(6)数据处理
基因分型数据经家系和人工校对后，用连锁分析软件LINKAGE(Ver.5.11)的MLINK程序计算每个标记位点的两点LOD值，参照Marneros的最近相似研究，设定重组率θ=0.000～0.400，遗传模式为常染色体显性遗传伴不完全外显，外显率90％，拟表型率1％，群体疾病基因型频率1／1000，根据各自的两点LOD值判断其连锁关系。
三、结果
1．遗传特征
男女患病率差异无统计学意义；瘢痕疙瘩致病基因在杂合状态下即可致病，父母之一发病，子女有50％的机率发病；家系中子女发病的父母之一大部分发病，小部分未发病作为基因携带者，共5个；多数家系子女发病的父母之一全发病；3个家系疾病性状连续传递，2个家系疾病性状间断传递，1个4代家系疾病性状已连续传递3代。
2．初始发病年龄
45例瘢痕疙瘩患者中，37例(82.22％)的初始发病年龄为16～25岁，正值中国人群的青春期。
3．首次发病诱因
45例瘢痕疙瘩患者中，38例(84.44％)首次发病诱因为痤疮和毛囊炎。
4．发病部位
45例瘢痕疙瘩患者中，发病于胸骨前21例(46.67％)、肩部+背部7例(15.56％)、面颈部5例(11.11％)、四肢5例(11.11％)、胸骨前+背部3例(6.67％)、面颈+胸背腹+四肢2例(4.44％)、面颈+肩部1例(2.22％)和腹部1例(2.22％)。
5．表现度的差异
六个瘢痕疙瘩家系中，不同家系或同一家系中不同患病成员瘢痕疙瘩的临床表型存在较大的差异。每个家系中先证者的临床表型最典型、病情最重，与同家系的其他患病个体存在较大的差异，而且，在同一家系中瘢痕疙瘩临床表型呈现出逐代减轻之趋势。
6．病变的形态特征
前胸部的瘢痕疙瘩多呈哑铃形，背部的多呈蝴蝶形，肩部和四肢的多呈螺旋形，面颈部的多呈条索状，腹部的则多呈不规则形。
7．具有对称发病倾向
45例瘢痕疙瘩患者中，29例(66.44％)呈现出以人体前后中线为中心轴的左右近似对称发病倾向。
8．连锁分析
在重组率θ=0.000～0.100时，微卫星标记D7S1818、D7S499、D7S494和D7S3046的两点LOD值都小于-2，否定疾病家系与这些标记的连锁关系；D10S1765和D10S1735的最大两点LOD值分别为1.743和1.218，支持疾病家系与D10S1765至D10S1735两标记间约1Mbp区域的连锁关系；D10S1562的最大两点LOD值为0.391，可以否定疾病家系与该标记的连锁关系；而D10S1687的所有两点LOD值均小于-2，则否定疾病家系与该标记的连锁关系。
四、结论
1．中国汉族瘢痕疙瘩家系的遗传模式符合常染色体显性遗传伴不完全外显，疾病表现度存在差异，临床表型呈现逐代减轻趋势。
2．中国人群瘢痕疙瘩家系以自发发病为主，初始发病年龄主要集中在16～25岁之间的青春期，好发于胸骨前和肩背部，病变形态因发病部位不同而异，并具有对称性发病倾向。
3．可以排除本研究中的内蒙古汉族瘢痕疙瘩家系易感基因位于染色体7p11上的可能。
4．中国汉族瘢痕疙瘩家系的易感基因可能位于染色体10q24.1上D10S1765与D10S1735两标记间约1Mbp的区域，并推测分布在该区域的PTEN基因和／或Fas基因可能是疾病家系的候选基因。
5．证实瘢痕疙瘩存在遗传异质性。
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 Methods
2.1 Subjects
These 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 Methods
2.2.1 Ascertainment for clinical phenotype of keloid and collection for keloid pedigrees
For 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 inheritance
To 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 primers
Referring 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 sequences
for the primers were obtained also, and the homologous primers were synthesesed then.
2.2.5 PCR amplifications, detections of amplified products and genotypings
These 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 processing
The 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. Results
3.1 Genetic characteristics
There 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 onset
Of 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 onset
Of the 45 affected family members, 38 (84.44%) cases were induced by acne and folliculitis.
3.4 Sites of display
Of 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 expression
The 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 features
Most 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 invasion
Of 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 analyses
Atθ=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. Conclusion
4.1 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.
瘢痕疙瘩是人类特有的一种创伤后病理性瘢痕愈合现象，它不同于其它瘢痕组织，具有过度生长、超过原伤口界限、并侵犯邻近正常皮肤组织、始终不退化和单纯手术切除后易复发等特点。瘢痕疙瘩不仅可继发畸形和功能障碍，影响美观，而且常伴有明显的瘙痒和疼痛症状，给患者造成极大的身心痛苦，是整形外科长期以来面临的重大难题之一。然而，因其病因至今仍不甚清楚，所以目前临床上尚缺乏特异性高的诊断方法和有效性强的治疗措施。可见，明确瘢痕疙瘩的病因将是准确诊断和有效治疗瘢痕疙瘩的根本途径。
关于瘢痕疙瘩的病因，国内外许多学者已经从组织学、病理学、生理学、免疫学和遗传学等方面开展了一系列的研究，提出了诸如胶原合成与降解失衡、细胞因子、成纤维细胞凋亡障碍、免疫反应和遗传学等许多学说，但这些学说都无法阐明瘢痕疙瘩发病的真正原因。瘢痕疙瘩大多散发发病，但具有一定的家族遗传倾向，Cosman等证实其家族性发生率约为3％，深肤色人种多发。有关瘢痕疙瘩的临床遗传学特征至今仍少有报道，而且其遗传模式仍未完全明了。Bloom通过对当时所报道的仅有一个五代瘢痕疙瘩意大利家系进行分析后认为它属于常染色体显性遗传，然而，Omo-Dare对所收集的瘢痕疙瘩小家系分析后认为它属于常染色体隐性遗传。Marneros等对14个瘢痕疙瘩家系进行临床遗传学方面的研究后发现，它们的遗传模式符合常染色体显性遗传伴外显不完全，且表现度存在差异，从幼童期到成年后期均可发病，大多数瘢痕疙瘩发生于胸部和肩部。由此可见，遗传是瘢痕疙瘩发病的主要因素，患病率和临床表型存在显著的种族差异。明确临床遗传学特征、定位易感基因、筛查和克隆致病基因等将成为今后瘢痕疙瘩病因研究的主要方向。
最近，Marneros等选择微卫星标记通过全基因组扫描和连锁分析对一个瘢痕疙瘩日本家系和一个非洲裔美国人家系进行易感基因的定位研究后发现，日本家系与染色体2q23连锁，非洲裔美国人家系与染色体7p11连锁，并认为位于染色体2q23上153cM(152Mbp)处的一个转录为肿瘤坏死因子-a抑制蛋白6(TNFAIP6)基因和位于染色体7p11上76cM(55Mbp)处的表皮生长因子(EGF)受体基因分别是日本家系和非洲裔美国人家系的一个候选基因，但是对这两个候选基因的外显子、内含子与外显子的拼接点及启动子序列进行测序，并未发现突变或与疾病相关的多态性存在，因此，认为可能在上述这两个候选基因位点附近其它基因的突变才导致对瘢痕疙瘩的易感。另外，他们还报道了对一个有10人发病的非洲裔美国人中等大小的瘢痕疙瘩家系进行同样的研究，却没发现该家系与染色体2q23和7p11存在连锁关系，提示至少有第三个瘢痕疙瘩易感基因位点存在，说明瘢痕疙瘩存在遗传异质性，这与临床上观察到不同家系瘢痕疙瘩病情不一的现象相一致。该研究首次提出了存在瘢痕疙瘩易感基因位点的遗传学证据，对进一步识别和定位瘢痕疙瘩的易感基因具有指导意义。由于瘢痕疙瘩的发病存在显著的种族差异，中国人群瘢痕疙瘩家系的临床遗传学特征和易感基因位点是否与Marneros等的研究发现相似，目前尚不清楚。为此，我们收集了六个中国汉族瘢痕疙瘩大家系进行这方面的研究，这些家系规模大、发病人数多，能较好地满足遗传病研究对家系的要求，对中国汉族人群瘢痕疙瘩家系的遗传学方面研究，具有较好的代表性。我们的研究发现，这些中国汉族人群瘢痕疙瘩家系的遗传模式也符合常染色体显性遗传伴外显不完全，且表现度存在差异。瘢痕疙瘩临床表型和遗传模式的确定为进一步开展其易感基因的定位研究奠定了良好的基础。
按照课题研究计划，由另一位博士研究生首先选择其中1个5代发病和1个4代发病的家系作为研究对象，也采用微卫星标记，通过连锁分析，否定了这两个中国汉族瘢痕疙瘩家系与染色体2q23的连锁关系。因此，本研究从上述那个5代发病家系中选择具有较高遗传学研究意义的32名成员作为研究对象，在Marneros等研究发现的基础上，于染色体7p11及其周围选取已知的四个最大两点LOD值的微卫星作为遗传标记，通过连锁分析，也否定了这个中国汉族瘢痕疙瘩家系与染色体7p11的连锁关系。为此，我们又从分析家系与目前被认为和瘢痕疙瘩发病可能存在密切关系的基因所在的染色体区域的连锁关系之途径来识别和定位瘢痕疙瘩的易感基因。
由于瘢痕疙瘩所具有的独特生长特性及临床表现，因此它可被视为一种创伤愈合后形成的良性肿瘤。现已证明成纤维细胞是瘢痕疙瘩形成的功能性细胞，其增殖-凋亡调控的失衡是导致瘢痕疙瘩不断增生而且难以退化的细胞学基础。Fas基因属于NGF／TNF受体家族，其相应的配体及单克隆抗体与之结合后可诱导细胞凋亡。Fas介导的凋亡被认为在成纤维细胞的凋亡中发挥着重要的作用。Fas基因位于染色体10q24.1上，是全长36kb的单拷贝基因。我们采用候选基因克隆策略来识别和定位瘢痕疙瘩易感基因，即假定Fas基因为导致瘢痕疙瘩易感的一个候选基因，选择位于10q24.1上Fas基因及其周围共约10Mbp的染色体区域内已知的与细胞凋亡障碍或肿瘤发生有关的基因(即Fas、PTEN、BMPR1A和DNTT基因)紧邻的4个微卫星作为遗传标记，通过连锁分析判断家系与这些微卫星位点之间是否存在连锁关系，以期识别和定位瘢痕疙瘩的易感基因，为进一步的致病基因相关研究提供理论依据。
二、材料与方法
1．研究对象
用于临床遗传学研究的六个中国人群瘢痕疙瘩家系中，来自辽宁2个、内蒙古、河北、福建和黑龙江各1个，均为汉族人群，家系之间无亲缘关系，家系成员无与汉族以外人种婚配史，无Rubinstein-Taybi和Goeminne综合症(伴发有瘢痕疙瘩)。在这六个家系中，5代家系1个，4代家系3个，3代家系2个；5代发病家系1个，4代发病家系2个，3代发病家系2个，2代发病家系1个；家系成员185个，男94个，女91个；发病45个，男18个，女27个，未发病的基因携带者5个。用于定位瘢痕疙瘩易感基因的家系来自内蒙古，5代发病，家系成员63个，男33个，女30个；发病14个，男7个，女7个；未发病的基因携带者3个；从该家系中选择具有较高遗传学研究意义的32名成员作为研究对象。本研究事先得到相关审核部门的同意，所有参与研究的家系成员均签署了知情同意书。
2．方法
(1)瘢痕疙瘩临床表型的确定和家系的收集
由2名对瘢痕疙瘩诊断和治疗具有丰富临床经验的整形外科医师，根据瘢痕疙瘩临床诊断标准(即瘢痕超过原损伤范围并向周围正常皮肤侵犯，或瘢痕病程超过1年仍无自发消退征象，或术后瘢痕复发者)把家系成员分为瘢痕疙瘩发病者和未发病者，对所有发病和未发病的家系成员进行详细的病史询问、全面的体格检查和准确的临床诊断，并对瘢痕疙瘩发病部位照相取证，建立个人临床信息资料档案(包括已故和无法到访的成员)。采集那个5代发病家系中具有较高遗传学研究意义的32名成员的外周静脉血供进一步的分子遗传学研究，并对部分患者的瘢痕疙瘩组织取活检行组织病理学检查以确诊，遵照赫尔辛基宣言准则使用这些人体组织进行相关的研究。
(2)绘制家系系谱图和分析遗传模式
根据收集的临床资料，绘制家系系谱以研究瘢痕疙瘩的遗传模式。虽然系谱包括未发病的儿童和青少年，但在分析遗传模式时予除外，因为随着年龄的增加，他们可能出现瘢痕疙瘩临床表型。未发病的家系成员其父母之一发病且至少有1个子女发病则称他或她为肯定携带者。
(3)提取基因组DNA
抽取每位家系成员的外周静脉血2ml(EDTA抗凝)，采用经典的酚氯仿法提取基因组DNA。
(4)选择微卫星标记和合成引物
通过查阅最近的相关文献和生物信息学数据库，在染色体7p11及其周围共18.8Mbp区域内选取4个已知的最大两点LOD值的微卫星和在Fas基因所在的染色体区带10q24.1及其周围共10Mbp的区域内选择4个与细胞凋亡障碍有关的已知基因相邻的微卫星作为多态遗传标记，用于定位瘢痕疙瘩易感基因研究；并查阅这些微卫星标记的上下游引物序列，以合成相应的引物。
(5)微卫星标记PCR扩增、产物检测和基因型分型
PCR扩增在PE9700型PCR反应仪中进行，产物经Genescan(Ver.3.11)和Genotyper(Ver.3.7)软件处理后根据检测片段大小进行基因型分型。
(6)数据处理
基因分型数据经家系和人工校对后，用连锁分析软件LINKAGE(Ver.5.11)的MLINK程序计算每个标记位点的两点LOD值，参照Marneros的最近相似研究，设定重组率θ=0.000～0.400，遗传模式为常染色体显性遗传伴不完全外显，外显率90％，拟表型率1％，群体疾病基因型频率1／1000，根据各自的两点LOD值判断其连锁关系。
三、结果
1．遗传特征
男女患病率差异无统计学意义；瘢痕疙瘩致病基因在杂合状态下即可致病，父母之一发病，子女有50％的机率发病；家系中子女发病的父母之一大部分发病，小部分未发病作为基因携带者，共5个；多数家系子女发病的父母之一全发病；3个家系疾病性状连续传递，2个家系疾病性状间断传递，1个4代家系疾病性状已连续传递3代。
2．初始发病年龄
45例瘢痕疙瘩患者中，37例(82.22％)的初始发病年龄为16～25岁，正值中国人群的青春期。
3．首次发病诱因
45例瘢痕疙瘩患者中，38例(84.44％)首次发病诱因为痤疮和毛囊炎。
4．发病部位
45例瘢痕疙瘩患者中，发病于胸骨前21例(46.67％)、肩部+背部7例(15.56％)、面颈部5例(11.11％)、四肢5例(11.11％)、胸骨前+背部3例(6.67％)、面颈+胸背腹+四肢2例(4.44％)、面颈+肩部1例(2.22％)和腹部1例(2.22％)。
5．表现度的差异
六个瘢痕疙瘩家系中，不同家系或同一家系中不同患病成员瘢痕疙瘩的临床表型存在较大的差异。每个家系中先证者的临床表型最典型、病情最重，与同家系的其他患病个体存在较大的差异，而且，在同一家系中瘢痕疙瘩临床表型呈现出逐代减轻之趋势。
6．病变的形态特征
前胸部的瘢痕疙瘩多呈哑铃形，背部的多呈蝴蝶形，肩部和四肢的多呈螺旋形，面颈部的多呈条索状，腹部的则多呈不规则形。
7．具有对称发病倾向
45例瘢痕疙瘩患者中，29例(66.44％)呈现出以人体前后中线为中心轴的左右近似对称发病倾向。
8．连锁分析
在重组率θ=0.000～0.100时，微卫星标记D7S1818、D7S499、D7S494和D7S3046的两点LOD值都小于-2，否定疾病家系与这些标记的连锁关系；D10S1765和D10S1735的最大两点LOD值分别为1.743和1.218，支持疾病家系与D10S1765至D10S1735两标记间约1Mbp区域的连锁关系；D10S1562的最大两点LOD值为0.391，可以否定疾病家系与该标记的连锁关系；而D10S1687的所有两点LOD值均小于-2，则否定疾病家系与该标记的连锁关系。
四、结论
1．中国汉族瘢痕疙瘩家系的遗传模式符合常染色体显性遗传伴不完全外显，疾病表现度存在差异，临床表型呈现逐代减轻趋势。
2．中国人群瘢痕疙瘩家系以自发发病为主，初始发病年龄主要集中在16～25岁之间的青春期，好发于胸骨前和肩背部，病变形态因发病部位不同而异，并具有对称性发病倾向。
3．可以排除本研究中的内蒙古汉族瘢痕疙瘩家系易感基因位于染色体7p11上的可能。
4．中国汉族瘢痕疙瘩家系的易感基因可能位于染色体10q24.1上D10S1765与D10S1735两标记间约1Mbp的区域，并推测分布在该区域的PTEN基因和／或Fas基因可能是疾病家系的候选基因。
5．证实瘢痕疙瘩存在遗传异质性。
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 Methods
2.1 Subjects
These 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 Methods
2.2.1 Ascertainment for clinical phenotype of keloid and collection for keloid pedigrees
For 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 inheritance
To 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 primers
Referring 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 sequences
for the primers were obtained also, and the homologous primers were synthesesed then.
2.2.5 PCR amplifications, detections of amplified products and genotypings
These 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 processing
The 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. Results
3.1 Genetic characteristics
There 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 onset
Of 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 onset
Of the 45 affected family members, 38 (84.44%) cases were induced by acne and folliculitis.
3.4 Sites of display
Of 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 expression
The 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 features
Most 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 invasion
Of 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 analyses
Atθ=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. Conclusion
4.1 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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