| Background & ObjectsKeloid, a real puzzle in plastic surgery, is a usual pathological scar in clinics. Though quite a number of researches have been reported, the pathogenesis of keloid is still unknown. Recent studies showed that keloid was the combined effect of genetic susceptibility and exogenous factors, so it is essential for keloid diagnosis and treatment to locate the keloid-susceptible gene(s). Previous studies had almost focused on sporadic cases, and the results were not typical and consistent for genetic variance and different surroundings, so they showed no specific conclusions. Pedigree, for its stable genetic background and typical phenotype, is the optimal object for discovering the susceptible gene(s), so collecting samples of affected pedigree to perform genetic analysis and related molecular genetic experiments probably will make a great breakthrough in keloid pathogenesis.Keloid pedigree appeared infrequently in clinic, and it was quite difficult to collect the whole samples of an affected pedigree. Furthermore, a single collection was not sufficient for a long-period study series, and repeated sampling was more difficult for scattered residence, poor cooperation and considerable expense. EBV (Epstein-Barr virus) is a B lymphocyte-tied herpes virus and can specifically transform B lymphocytes to LCL (lymphoblastoid cell lines) , which have perpetual proliferative talent. Based on this characteristic, an immortalized lymphocyte cell bank of pedigree will be establish with EBV transforming technology, as a result, the genetic information of the pedigree could be permanently preserved and sufficient samples could be used in a long-term research.STR (short tandem repeats) are widely distributed in eukaryote genome, they are considered to be a newly appeared genetic markers for their highly polymorphism. Currently, performing STR-related molecular experiments and linkage analysis of pedigrees to find the disease related STR markers is the optimal method to locate the susceptibility gene(s).In this paper, we collected the whole blood samples and clinical data of a Han Chinese keloid pedigree, and then established the LCL banks of important members with EBV transforming technology. To preliminarily locate the susceptible locus to keloid in this pedigree, we picked 7 STR markers and performed linkage analysis to human chromosome 15q22.31-q23&18q21.1, where SMAD genes lies in.Materials & Methods1. We collected full clinical data and peripheral blood samples of a Han Chinese keloid pedigree in northeast China and make a preliminary genetic analysis.2. Establishing the LCL banks of the keloid pedigreeWe chose 28 blood samples including the proband and a 4-generation family to establish the LCL banks with two methods.One was with fresh blood, the lymphocytes were isolated from blood samples and mixed with prepared EBV and CyA to be cultured in incubator at 37℃, 5%CO2; We passaged 4～5 times after the lymphocytes being successfully transformed, and then froze the cell strains in liquid nitrogen. In 3 months, the frozen cells were revivified to be cultured for comparison.The other way was with frozen blood sample. We picked 6 samples from all frozen blood, revivified them and mixed with prepared EBV to be cultured in routine.We performed analysis of the chromosomal karyotype of the lymphocytes surrounding the transforming process and after revivification.3. Linkage analysis of keloid susceptible locus in this pedigreeThirty two blood samples in this pedigree were picked for genetic mapping. Seven microsatellite markers spanning the critical regions on chromosomes 15q22.31-q23 and 18q21.1 were genotyped, and the DNA extracted from each sample was amplified for these markers. The PCR products of them were scanned at ABI PRISMTM 310 Genetic Analyzer, and the eligible genotyping data were used for linkage analysis. We calculated the two-point LOD scores with MLINK program of Linkage5.11 to judge the linkage level.Results and Discussion1. We fully collected clinical data and peripheral blood samples of a 5-generation keloid pedigree in northeast China. This keloid pedigree was autosomal dominant inheritance with incomplete penetrance, and the expressivity was various in different members.2.27 immortal lymphoblastoid cell lines of this pedigree were obtained successfully, and all of the immortal lymphoblastoid cell lines were revivificated after being frozen in liquid nitrogen. All the results of the chromosome karyotype analysis showed the LCLs kept genetic stability. We achieved higher success rate in transforming process with the fresh blood than the frozen blood, and the former method was the optimal one for establish LCL banks, while the latter was possibly a complementary one.3. Among the 7 selected markers, the two point LOD scores of 5 ones (D15S108,D15S216,D15S534,D18S363,D18S846) were less than -2 atθ=0, so they were excluded the linkage to the disease locus; The scores of others were either more than 1 atθ=0.05 & 0.10, which showed some linkage to the susceptibility locus. It needs to be confirmed by further research in the future.Conclusions1. We fully collected clinical data and peripheral blood samples of a Han Chinese keloid pedigree, which is the largest one reported to date. This keloid pedigree was autosomal dominant inheritance with incomplete penetrance and consistent with previous reports.2. We firstly used EBV transforming technology to preserve the DNA of the keloid pedigree and established the immortal LCL banks of an affected pedigree. This technology was a useful way to preserve the genetic information of more keloid pedigrees in the future. We preferred the method with fresh blood in transforming process.3. Our results of STR linkage analysis firstly provided some evidence that keloid susceptibility gene of this Chinese pedigree located on chromosome 18q21.1; SMAD2 and PIAS2 may be the candidates. We proposed to collect samples of more keloid pedigrees for genome scan and linkage analysis to locate and clone the susceptibility gene to keloid in our Chinese people. |
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