Study On Dhtkd1^Tyr486* Knock-in Mouse Model And Gene Diagnosis Of A Cone-Rod Dystrophy Family

PART 1 Study of Dhtkd1 ^Tyr486* knock-in mice modelCharcot-Marie-Tooth disease?CMT?is a kind of most common inherited neurological disorders.In 2009,our research group collected a large pedigree with CMT2 disease,by using whole genome scanning and linkage analysis,we successfully identified a nonsense mutation in exon 8 of DHTKD1?dehydrogenase E1and transketolase domain containing 1?gene[c.1455T>G?p.Tyr485*?],and named this type of CMT as CMT2Q.We constructed Dhtkd1 ^Tyr486*knock-in mice model by genetic engineering method,and performed detailed phenotypic analysis to evaluate the mouse model.However,the mice model did not present the serious symptoms as human affected with CMT2Q as we expected.The RNA chip had been taken to find out differentially expressed genes between mutant homozygotes and wild type mice.Result showed that homo mice's Dgkg gene expression increased compared to wt mice?fold changed>2,P-value<0.01?.Dgkg encode diacylglycerol kinase gamma,which phosphorylate diacylglycerol to phosphatidic acid.We predicted that homo mice might through glycerolipid metabolism to make up the poor energy production due to the lack of Dhtkd1.Ataluren?PTC124?as a nonsense suppression drug promotes translation through premature stop codons.Our study is designed to investigate whether PTC124 can promote readthrough of the Dhtkd1 ^Tyr486*mutation.After PTC124-treamemt,the mouse embryonic fibroblast from homo Dhtkd1 ^Tyr486*knock-in mice showed that the expression of Dhtkd1 was up-regulated?p<0.05?.PART 2 Identification of the mutant gene in a large pedigree with cone-rod dystrophyHereditary retinopathy is not uncommon in ophthalmology.We found a large family with autosomol dominant hereditary retinopathy.All affected members were taken by using interrogation and ophthalmological examinations.However,it was still hard to make a definite diagnosis to this family since the symptoms among different hereditary retinopaties are very alike.To make gene diagnosis and identify the mutant gene and clinical manifestation in this family,the exon-sequencing of 372 ophthalmic disease-associated genes was done with Illumina Hi Seq 2000 platform.Ten highly suspicious mutation hotspots were chosen to confirm in all family members with Sanger sequencing.All affected members presented very progressive cone-rod dystrophy,and clinical features showed onset earlier and serious symptoms.A missense mutation in exon 3?c.238G>A?of CRX gene was identified,resulting in an amino acid change from glutamic acid to lysine?p.E80K?.It is the first report of CRX mutation?p.E80K?pedigree in Mongolian people.At the same time,the study suggested that the clinical manifestations in p.E80 K were more serious than that of p.E80 A and p.E80 Q.