| In the course of evolution,skin color changes are the most typical adaptive phenotypic characteristics,and the diversity of skin color is very high among people living in different environments and climates.Through comparative genomics studies,it has been found that the skin color of the ancestors of European and Asian populations is independent evolutionary event,and there are significant differences in the genes that are positively selected.So far,most of the genetic studies of pigment-related phenotype are carried out in European populations,but the causes and mechanisms of pigment-related phenotypic differences may be different in different populations.Using the genome-wide microarray data in Taizhou cohort,we conducted a genome-wide association analysis with the pigment-related phenotype.It was found the OCA2 gene rs74653330 site mutation was associated with the light skin color.In order to further verify the effect of this mutation site on the pigment-related phenotype,this experiment used the CRISPR-Cas9 method to construct the melanocyte model for later functional verification experiments.In this experiment,the recombinant p X459 plasmids with g RNAs sequences were successfully constructed,and the gene editing efficiency of g RNAs was preliminarily determined.The transfection condition was determined on the A375 melanin cell line,but the A375 cells had three bodies on the site that we needed to edit,and then the A2058 melanin cell line was used for the experiment.In the process of the experiment we have purchased A2058 melanin cell line from different cell libraries,and all have mycoplasma contamination.This experiment failed to get positive mono-clones,but established the experimental system for building the directed mutation melanocyte model on the OCA2 gene rs74653330 SNP site.This will be guiding significance to the mycoplasma-free cells transfection,sorting mono-clones,and screening positive cells.Nystagmus is an eye disease characterized by repetitive,rhythmic and involuntary movement.Due to consecutive eye movement,visual function is significantly reduced,but the degree of visual impairment varies.The disease can be secondary to other visual or neurological diseases,or can be an independent genetic trait of congenital nystagmus(CN).Congenital nystagmus usually occurs at birth or in the first few months of life,with an estimated annual incidence of 1/20,000.So far,FERM structure domain 7 gene(FRMD7)and G protein coupled receptor 143 gene(GPR143)have been identified as the pathogenic gene of CN.We scanned with all four individuals’(three male patients and one female carrier)exons which were in a CN family of six generations,and found 5 new SNP loci on the X chromosome which may be associated with the disease phenotype.They respectively are rs146443503,rs141478957,rs3810704,rs2233695 and rs2205912,which has never been reported before.In order to verify the functional relevance of these five SNP sites,the re-sequencing of relevant SNP sites was carried out in this CN family.A genealogical analysis of the sequencing results was conducted to determine the genetic approach of CN in this family and to explain the relationship between SNP sites genotype and CN phenotype.Finally,only male patients were found in this CN family,and the females were only carriers,and the obvious X-linked recessive inheritance law was reflected.The correlation between phenotype and SNP sites mutation can be found after analyzing sequencing results which include the information of genetic transmission in this family.Pathogenic gene research is usually limited to FRMD7 and GPR143 before,there is no doubt that what the study found enriches the CN molecular mechanism explanation,and for the possible genetic counseling,prenatal diagnosis and gene replacement therapy provide a certain reference. |
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