Neonatal Screening For Primary Immunodeficiency Diseases And Abtacept Is Effctive In Chinese Patients With LRBA And CTLA4 Deficiency

PART I NEONATAL SCREENING FOR PRIMARY IMMUNODEFICIENCY DISEASESObjective:Our study takes advantage of the current neonatal screening system,which is based on the detection of dry blood spots,and we intends to set up a pilot neonatal screening system for primary immunodeficiency diseases in China.By detecting the copy numbers of T/B cell antigen receptor excision circles(TRECs/KRECs)using real-time quantitative PCR,we can provide a normal reference range of TRECs/KRECs values for different gestational ages and birth weights of newborns in China.Meanwhile,it will contribute to the early detection,diagnosis and treatment of PID patients.Methods:In this study,we established a triplex real-time quantitative PCR method for the combined detection of TRECs,KRECs,and the housekeeping gene RNase P.We tested a total of 5039 newborns and compared this triplex RT-qPCR with the Roche PCR methods.Results:The triple real-time PCR method used in this research has the advantages of simple operation,high reproducibility,wide linear range,and high resolution.Compared with the Roche TRECs and KRECs detection methods,this method is available for absolute quantitation of TRECs and KRECs,and it is more sensitive for the detection of KRECs.We tested a total of 5039 newborns in this study.The average gestational age of newborns was39+2 weeks,of which 174 were premature and 10 were post-term.The average birth weight of newborns was 3310 g,of which 1 had very low birth weight,111 had low birth weights and 205 were macrosomia.The average concentration of DNA from the dried blood spots was 11.4(3.4-23.3)ng/?L.The mean and median number of TRECs were 288 and 255 copies/?L whole blood respectively,and the mean and median number of KRECs was 187 and156 copies/?L whole blood respectively.Two samples had low concentration of DNA.After re-spotting and re-testing,RNase P was still below 2000 in one sample,with normal numbers of TRECs and KRECs.There were seven neonates with abnormal TRECs in the first DBS,and four of them had normal results after the second DBS.Of the 3 neonates whose TRECs were still abnormal after the second DBS,1 had a TRECs number of 0 and was recalled.There were 52 neonates with abnormal KRECs in the first DBS,and 46 of them had normal results after re-punch test.Of the 6 neonates whose KRECs were still abnormal after re-punching,one case has been recalled,and the rest could not be followed up in time due to the 2019coronary pneumonia outbreak.The copy number of TRECs in preterm infants was lower than that of full-term infants,and there was no significant difference in TRECs,KRECs,and RNase P among neonates with different birth weights.Lymphocyte classification and immunophenotyping tests were performed on the neonate whose TRECs was 0.The results showed that the number and proportion of CD3~+T cells,CD8~+T cells,and CD4~+T cells were significantly reduced,and most of the CD8~+T cells and CD4~+T cells were TEMRA and memory cells.The number of CD19~+B cells and NK cells was normal.The immunological features in this neonatal was similar to that in T~-B~+NK~+SCID patients.Conclusion:Our study successfully established a triple real-time quantitative PCR method for the combined detection of TRECs,KRECs,and RNase P.This method has the advantages of simple operation,high reproducibility,wide linear range,high resolution and low cost,which can be applied to the neonatal screening of PID.In this study,the reference range of neonatal TRECs and KRECs was determined for the first time in China.A newborn with a TRECs number of 0 and an immune phenotype consistent with T~-B~+NK~+SCID was screened.Therefore,this study provided technical support and experience to promote domestic PID newborn screening.PART ? ABTACEPT IS EFFCTIVE IN CHINESE PATIENTS WITH LRBA AND CTLA4 DEFICIENCYObjective: To analyze the clinical,immunological features and treatments of two patients with LRBA deficiency and one with CTLA4 deficiency.In order to improve the clinical diagnosis and treatment of these patients,the long-term efficacy of abatacept therapy was studied.Methods: Two patients with LRBA deficiency and one with CTLA4 deficiency were enrolled in this study.The clinical manifestations and detailed treatments including steroids,IVIG,immunosuppressants as well as abatacept were collected for each patient.Whole-exome sequencing was performed on genomic DNA of three patients.Mutations in the LRBA and CTLA4 gene were verified by Sanger sequencing for patients and their parents.We also evaluated Treg cells,CTLA4 expression and c Tfh cells using flow cytometry.Results: Here we reported three Chinese patients with LRBA and CTLA4 mutations,they all presented with chronic diarrhea,hypokalemia,organomegaly,recurrent infections,and hypogammaglobulinemia.Reduced Treg cells,CTLA4 expression and increased percentage of c Tfh cells were revealed in these patients.Although steroids and immunoglobulin therapy were given,the enteropathy was persistent.Therefore,abatacept treatment was provided to the patients.They showed a marked improvement of enteropathy and gastrointestinal endoscopy showed alleviated inflammatory lesion and follicular hyperplasia.Furthermore,the frequency of c Tfh cells was reduced after abatacept therapy.Conclusion: Targeted therapy with abatacept is a promising treatment modality for patients with LRBA and CTLA4 deficiency.The findings also suggest that the frequency of c Tfh cells could serve as a marker for tracking disease activity and the response to abatacept therapy.