Establishment Of Immortal Lymphoblastoid Cell Lines Of The Goldenhar Syndrome Cases And Preliminary Study Of Disease-causing Gene Of A Goldenhar Syndrome Pedigree

Part I Establishment of immortal lymphoblastoid cell lines of a Goldenhar syndrome pedigree and sporadic casesObjectiveTo establish the immortalized lymphoblastoid cell lines of a family and sporadic cases of Goldenhar syndrome.MethodsThe peripheral blood samples of a Goldenhar syndrome family and family members of 4 other sporadic cases were collected. B lymphocytes were transformed by Epstein-Barr virus to establish immortalized cell lines and cyclosporine A was used to inhibit T lymphocytes. Genetic stability was detected by karyotype analysis after post-transformed cell freezing and thawing. The transformation of B lymphocytes was observed under light microscope. The recovery success rates of the immortalized cell lines were calculated and the chromosomes were analyzed through G-banding stain.ResultsAfter about 1 week of culture, it was seen under the inverted microscope that B lymphocytes obviously enlarged, with various shapes, assemble and there were obvious cytoplasmic prominences around the cells. After about 15 days, a few white cell colonies were seen by the naked eyes. About 1 month, the cells proliferated and became the dense colonies.31 immortalized cell lines were established, including 8 cases and 23 family members, and the success rate of the former was 100% and that of the latter was 95.8%(23/24). The recovery success rate of all the immortalized cell lines was 100%. The genetic integrity of the cell lines was stable by karyotype analysis through G-banding stain, and their karyotypes were normal. ConclusionThe karyotypes of the patients and their family members are normal. The cell lines of Goldenhar syndrome established in this study can be useful for exploring the mechanism in the future research. PartⅡExclusive mapping of the Known locus 14q32 and mutation detection of EYA1 in a Goldenhar syndrome pedigreeObjectiveTo explore the pathogenic cause of Goldenhar syndrome, map the known locus 14q32 linked with Goldenhar syndrome, and detect the mutation of EYA1 gene in the Goldenhar syndrome cases.Methods(1) Genomic DNA was extracted from peripheral blood samples.4 microsatellite markers located in 14q32 were choosen and linkage analysis was performed using short tandem repeat (STR) polymorphism markers by using MLINK to calculate two-point (logarithm [base 10] of odds) lod scores.(2) Four patients and two suspects in a Goldenhar syndrome pedigree and four sporadic patients were recruited. The exons3-18 of EYA1 gene were analyzed by polymerase chain reaction and direct sequencing.Results(1) Four microsatellite markers were not cosegregated with the phenotype in this family. LOD scores showed negative values. Two-point lod score linkage analysis excluded the known genetic loci and candidate mutant genes in all patients.(2) We found 3 SNPs in 8 patients and 2 suspects.Conclusion(1) The results of hyplotype and linkage analysis suggest that the candidate mutant genes of this Chinese family were not located in 14q32, suggesting that Goldenhar syndrome appears to be have genetic heterogeneity.(2) EYA1 gene mutation can be excluded in a Goldenhar syndrome, suggesting that there is a new gene associated with the disease. PartⅢDisease-causing genes mapping of a Goldenhar syndrome pedigreeObjectiveTo map the locus of disease-causing genes linked with Goldenhar syndrome, colne, and find out the molecular mechanisms of the disease in this family.MethodsDNA samples of the Goldenhar syndrome pedigree were genotyped on the Illumina Sentrix Human CNV370-Quadv3-0 chip. Parametric multipoint linkage analysis was performed using the MERLIN software under the assumptions of autosomal dominant inheritance with 80% and 99% respectively penetrance, a disease allele frequency of 0.1%, and equal SNP allele frequency(50%). STR markers on the related locus were choosen to check the results.Results(1) Maximal LOD score 1.21 was obtained that it was mapped on 15q26.2-q26.3 and 22q11.1-q11.22. In non-parametric linkage analysis, LOD scores were over 2 mapping on a few chromosomes, such as 4p15.32,8p12,12q24.31,14q22.3-q31.1,15q26.2-q26.3 and 22q11.1-q11.22。(2) In two-point parameters linkage analysis, LOD scores 1.2 and 1.19 were obtained near D14S258 and D22S539 respectively.ConclusionLocalization of candidate genes in a Goldenhar syndrome pedigree could be on 22q11.1-q11.22.