Mutation Of TWNK Gene Is One Of The Reasons Of Runting And Stunting Syndrome Characterized By MtDNA Depletion In Sex-linked Dwarf Chicken

Runting and stunting syndrome(RSS)in chickens is commonly known as “frozen chicken”.This condition is characterized by lower body weight,slow growth and widely occurs in sex-linked dwarf(SLD)chickens.The clinical symptoms of RSS chickens are similar to human mitochondrial diseases.Therefore,we speculate that this type of RSS may be caused by mitochondrial dysfunction in chicken.First,we used RSS chicken,SLD chicken and normal chicken for liver pathological analysis,and determined the enzyme activity of OXPHOS complexes and mitochondrial function in liver.Histopathology studies demonstrated that the hepatocytes of RSS chickens displayed mitochondrial damage.Hematoxylin and eosin and Oil Red O staining of the chicken livers confirmed that there was no inflammatory response,and an abundance of lipid vacuoles was observed in the hepatocytes.We also observe abnormal mitochondrial morphology in the RSS chickens and SLD chickens by transmission electron microscopy(TEM)analysis.Meanwhile,we found that reduced enzymatic activity of OXPHOS complexes and impaired mitochondrial function both in RSS and SLD chickens.Then,we interfered with the expression of GHR in DF-1 cells.We found that the expression of key regulators of mitochondrial biogenesis and mitochondrial DNA(mt DNA)-encoded OXPHOS genes were downregulated and accompanied by reduced enzymatic activity of OXPHOS complexes in GHR knockdown cells.Then,we found that mitochondrial function was impaired in GHR knockdown cells.In addition,we also studied the morphology and structure of mitochondria in GHR knockdown cells by TEM and Mito Tracker staining.At last,we discussed the molecular mechanism of RSS.Here,we identified a homogeneous mutation c.409G>A(p.Ala137Thr)of TWNK gene in RSS chickens from strain N301.Bioinformatics investigations supported the pathogenicity of the TWNK mutation that is located on the linker region of Twinkle primase domain and might further lead to mt DNA depletion in chicken.In addition,we also found that overexpression of the TWNK wt can increase mt DNA content in LMH and DF-1 cells,whereas overexpression of the TWNK A137 T can cause mt DNA depletion in LMH and DF-1 cells.In summary,the GHR gene is essential for chicken mitochondrial function in vivo and in vitro.Further,the TWNK gene missense mutation c.409G>A(p.Ala137Thr)might lead to the emergence of RSS characterized by mt DNA depletion on the basis of mitochondrial dysfunction caused by GHR mutation.We argued that the mutation of TWNK gene is one of the reasons of runting and stunting syndrome characterized by mt DNA depletion in sexlinked dwarf chicken.However,the mechanism of interaction between the GHR mutation and TWNK mutation remains unknown in chicken.