Phenotypic Analysis Of Dhtkd1^Tyr486* Knock-in Mouse Model And Pathogenic Mechanism Of Hereditary Protein C Deficiency

Part 1 Phenotypic analysis of Dhtkd1 Tyr486* knock-in mouse modelCharcot-Marie-Tooth disease(CMT)is a kind of most common inherited neurological disorders. It`s morbidity in human population is about 1/2500. In the previous work, our 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 E1 and transketolase domain containing 1) gene [c.1455T>G(p.Tyr485*)]. In vitro experiments found that nonsense mutation in DHTKD1 had negative effect of energy production in mitochondria, which considered as the pathogenic mechanism of CMT2 Q. Therefore, we constructed Dhtkd1 Tyr486* knock-in mouse model by genetic engineering method, and performed detailed phenotypic analysis to evaluate the mouse model. We found the model with normal birth rate, Dhtkd1 decrased in heterzygotes and homozygotes in both m RNA and protein livel; ultrastructure of sciatic nerve in heterzygotes and homozygotes showed obviously myelin redundancy and mitochondria damage in axon. Ultrastructure of skeletal muscle showed mitochondrial vacuolation and muscle fiber dissolution. Behavioral tests detected sensory loss and motor dysfunction in knock-in mouse model. In conclusion, our study indicated that the Dhtkd1Tyr486* knock-in mouse model recapitulated some phenotypes of CMT2 Q. Although the mechanisms underlying CMT2 Q is still not clear, we demonstrate the important role of Dhtkd1 in maintaining the normal state of peripheral nerve system and metabolism, suggesting a close relationship between the two aspects during the progression of CMT2 Q.Part 2 Pathogenic Mechanism of Hereditary Protein C DeficiencyHereditary Protein C deficiency is a kind of inherited thrombophilia. It’s morbidity in human population is about 1/16000, mainly caused by genetic mutations in PROC gene. Currently, more than 270 mutations in PROC had been reported, among these mutations, missense mutations take a large proportion. We collected two patients with deep venous thrombosis, and diagnosed them as hereditary protein C deficiency through detailed clinical examination and coagulation assays. We found three mutations in PROC [c.541T>G, c.595C>T, c.740G>A] by performing direct sequencing. We constructed recombinant plasmids harbouring above-mentioned mutations, and transfected them into HEK293 cells, and found mutants were rapidly degraded; this phenomenon was rescued with the knockdown of UPF1, the core factor of nonsense-mediated m RNA decay pathway. Our data demonstrate that nonsense mutations in PROC cause a decrease in mutant m RNA levels by NMD rather than production of truncated proteins, implicating that the degradation of PROC mutant m RNA by NMD is an important mechanism in hereditary protein C deficiency patients with nonsense mutations.