The Negative Regulatory Mechanism Study Of ISG15 On Autoinflammatory Disease

Intracranial basal ganglia calcification is a neurological disease characterized by ectopic calcification in the basal ganglia and/or other parts of the brain.The disease in patients with a wide spectrum of manifestations of Parkinson,memory loss,epilepsy,mental disorders and headache and other symptoms and it has genetic heterogeneity that the genetic pattern is mostly autosomal dominant and the few are recessive.Intracranial calcification is also a well recognized feature of a group of Mendelian autoinflammatory diseases associated with upregulation of IFN-α/β signalling,including Aicardi-Goutieres syndrome（AGS）and spon-dyloenchondromatosis（SPENCD）,in particular.In our current research,We investigated a Consanguineous family with IBGC from of China.The probands showed seizures at an early age and die during an episode of epileptic seizures.The other two siblings,currently aged 11 and 13 years,have suffered seizures as well,But another young brother and parents are normal.Computed tomography（CT）scan showed that three children with epilepsy with basal ganglia calcification symptoms,unaffected family members do not have this symptom.To identify the disease gene of of Basal ganglia calcification in this family,we used sangersequencing and whole exome sequencing methods to identify the disease gene.Firstly,through Sanger sequencing,we found that there are no mutations in the three genes of IBGC:SLC20A2,PDGFRA,PDGFB in the family.And then we do the whole exome sequencing on the two patients and their healthy mother,we found that in the patients who have a nonsense homozygous mutation of ISG15 gene,while in the unaffected child and their parents carry the heterozygous mutation.Importantly,the mutation of ISG15 gene has not been reported in the internal WES data of the public database or other 1,500 individuals.The mutation(c.163C>T/163C>T;p.Gln 55*/Gln 55*is located in the exon II of the ISG15 gene,the Gln 55 in ISG15 is highly conserved during evolution.Sanger sequencing analysis in the patients and 100 normal individuals,showed that all the unaffected individuals did not carry the mutation.These results suggested us that ISG15 gene is candidate gene of IBGC.At the same time,our collaborator reported that ISG15 gene mutation associate with Mendelianus Mycoba-cterium susceptibility disease（MSMD）,the MSMD patients carry the homozygous mutation,These ISG15 gene mutations are c.379G>T/379G>T（p.Glu127*/Glul27*）and c.336₃37insG/336₃37ins.We ask our collaborator do the CT scan on the MSMD patients who carry the ISG15 mutation,CT scan results showed that the patients with basal ganglia calcification symptoms.Those results further confirmed that ISG15 is a new diseasegene of IBGC.We collaborate with the scientist in U.S.and France demonstrated that ISG15-deficient patients have unexpectedly elevated IFN-α/β immune responses in cytology,immunology,and clinically.Our research confirmed that lack of ISG15 in the human cells can inhibit the accumulation of USP18,a negative regulatory factor in the IFN-a/(3 signaling pathway,and can lead to enhanced and expanded response to IFN-α/β.Thus,human intra-cellular ISG15 not only plays a role in antiviral immunity,but also functions as a key negative regulator in the IFN-a/p immune response.For human,intracellular ISG15 is induced by IFN-α/β,not always acts as a substrate of isgylation-dependent antiviral immune response,but this time was used to ensure the USP 18-dependent regulation of IFN-α/β,thereby preventing IFN-a/p-dependent autoinflammation.In addition,in this project we demonstrated that silence the ISG15 gene in human microglia cells significantly increase the expression of IL-1β and IL-18.The further study showed that ISG15 maybe negatively regulate the expression of IL-1β through the NF-KB-inflammasome pathway.IL-1β is a proinflammatory factor,too much expression will induce inflammation,especially in the brain inflammatory response and neurode-generative diseases.And the clinical symptoms and cytological phenotypes of patients in our study are very similar to the inflammatory diseases:Aicardi-Goutieres syndrome and spondyloenchondrodysplasia.In conclusion,our study identified a new diseasegene ISG15 of IBGC,and then collaborated with scientists in U.S.and France decipher the molecular mechanism.After previous work published,we did further study and found that ISG15 is a potent negative regulator of secretion of IL-1β and IL-18.Our study provides a new theoretical basis for clinical diagnosis and an effective Drug target for the treatment of the basal ganglia calcification disease.