Identification Of Pathogenic Mutations In Deficiency Of Adenosine Deaminase 2 And Autoimmune Disease,Multisystem,Infantile-Onset,1

Part Ⅰ: Identification of Pathogenic Mutations in Deficiency of Adenosine Deaminase 2Objective This study took 1 case of autoinflammatory disease family as the research object,using whole-genome sequencing technology,real-time fluorescent quantitative PCR technology,polymerase chain reaction and Sanger sequencing technology to screen and identify pathogenic variants.This study provides a basis for the determination of the clinical diagnosis and treatment plan of the patient and enriches the gene mutation database of the diseaseMethods Firstly,we collected the clinical data of the proband and family members.Secondly,we collected the families’ peripheral blood to extract DNA.Then we used Sanger sequencing and whole-genome sequencing technology to screen out possible pathogenic variants in the data.Polymerase chain reaction and Sanger sequencing technology were used for verification in normal people.The patient is currently suspected of having deficiency of adenosine deaminase 2.Deficiency of adenosine deaminase 2 is inherited in an autosomal recessive manner,so other pathogenic variants need to be found.The peripheral blood RNA of patients and normal people of the same race was extracted and reverse transcribed into c DNA.The expression of ADA2 at the m RNA level was detected by real-time fluorescent quantitative PCR technology.We also used real-time fluorescent quantitative PCR technology to search the approximate range of copy number variations.At last we designed primers based on this range,and used Gap-PCR to find out the exact positions of the breakpoints on both sides of the copy number variations.Result 1.The patient in this study is a 21-year-old male,he developed a disease at 14 years old.The clinical manifestations were dizziness,unstable walking,repeated fever and limb weakness.MRI showed "abnormal signals in the right thalamus and brainstem",and chest CT showed "multiple lymph nodes can be seen in axilla on both sides".The patient’s hormone therapy has a good effect.2.The whole genome sequencing of the patient found that the patient’s ADA2 had a missense variant c.505C>G,and sanger sequencing detected c.505C>G missense mutation.3.Real-time fluorescent quantitative PCR suggested that: compared with the peripheral blood m RNA of the patient’s father and normal people of the same race,the expression level of ADA2 in the patient and his mother is half of normal people.Compared with the peripheral blood g DNA of the patient’s father and normal people of the same race,the expression level of exon 7 in ADA2 of the patient and his mother was half of normal people.The expression level of other exons of ADA2 at the g DNA level was similar to normal people.4.We used Gap-PCR to determine positions of the breakpoint: the positions of the breakpoints on both sides of the missing sequence are chr22:17,669,598 and chr22:17,668,824 respectively.5.Pathogenicity prediction: c.505C>G variant has not been reported in other public databases such as Ex AC and Thousand People Database.We took a functional prediction on Var Cards,software such as VEST3,CADD indicated that the mutant protein is damaging.Conclusions The patient in this subject has a compound heterozygous mutation in ADA2,which can be diagnosed as deficiency of adenosine deaminase 2.There are two pathogenic mutations,one is from the father(NM＿001282225: exon3: c.505C>G: p.R169G),the other is from the mother(c.17669228-404＿17669337+261del breakpoint).Multiple prediction softwares suggested that the mutant protein of p.R169 G is pathogenic.Part Ⅱ: Identification of Pathogenic Mutations in Autoimmune Disease,Multisystem,Infantile-Onset,1Objective In this study,a family with autoimmune disease,multisystem,infantileonset,1(ADMIO1)was used as the research object.High-throughput sequencing technology was used to screen the pathogenic variants of the family.We were aimed to explore the pathogenic mechanism and provide the necessary genetic basis for the family reproductive guidance.Methods The clinical data of the proband and family members were collected,then we got peripheral blood and extracted genomic DNA.We used whole exome sequencing to screen out candidate genes.Next,we judge the genetic mode of related diseases,and verify whether they are co-segregated in the family through sanger sequencing.The peripheral blood RNA of the family and normal people of the same race was extracted and converted into c DNA.The expression level of the candidate variants at the m RNA level was evaluated by real-time fluorescent quantitative PCR technology.Then we predicted the conservative type between species in UCSC and checked its frequency in EXAC,gnom AD and other databases.Finally,we used Mutation Taster,CADD,SIFT and other software to predict the pathogenicity of mutant protein.Result 1.The proband in this study was a 17-year-old-female,who became ill after 40 days of birth.The clinical manifestations were mainly red papules on the trunk,limbs,scalp accompanied by itching,running water and suppuration after scratching,desquamation after healing,Joint pain and swelling of a toe joint.Physical examination can find splenomegaly.Hormone therapy is effective.The father of the proband suffered from "seborrheic dermatitis",joint pain and low back pain.He was diagnosed as "large granular lymphocytic leukemia" in 2019;2.Whole exome sequencing results: The proband and his father had a missense mutation c.454C>T in the STAT3,which led to the variant p.Arg152Trp;3.Sanger sequencing results: the same variant was not detected in the normal members of the family and 200 healthy control volunteers;4.Pathogenicity prediction: The c.454C>T variant has been reported in an article in the HGMD database.The c.454C>T variant reported in their article can led to ADMIO1.Patients with the same variant that have been reported mainly present with autoimmune cytopenia and autoimmune thrombocytopenia,accompanied by type I diabetes,alopecia,pulmonary nodules,lymphadenopathy,and hepatosplenomegaly.Conclusion This study,as the first case study of ADMIO1 in China,elucidated the STAT3 c.454C>T pathogenic gene mutation in a family with ADMIO1 from the genetic level,which was the prenatal diagnosis for this family.Provide a basis.The clinical phenotype caused by the same variant STAT3(c.454C>T)was different,which suggested that clinicians should take consideration comprehensively when making diagnosis.The father of the proband was diagnosed with large granular lymphocytic leukemia,and studies had pointed out that gain-of-function mutations in the somatic STAT3 are related to large granular lymphocytic leukemia.Therefore,systematic treatment and regular monitoring is required for the proband to provide help for delaying disease progression.