Utility Of Next-generation Sequencing Technologies For The Efficient Genetic Resolution Of Rare Haematological Disorders In Childhood

Background: There're some rare haematological disorders,such as Juvenile myelomonocytic leukemia(JMML),Diamond Blackfan anemia(DBA)and Dyskeratosis congenital(DC),which have complex genotypic and phenotypic variations.Objective: To complete the genomic landscape of JMML,investigate the genotype-phenotype correlation,explore the role of mutation in the transfamation of JMML to acute leukemia.To investigate the genetic features of DBA and DC in Chinese pediatric patients.Methods: We sequenced samples from patients with JMML,DBA,DC screening a panel by NGS.Seven of these patients also had serially acquired samples from transformation to acute myeloid leukemia(AML)available for WES.Results: In addition to canonical Ras pathway driver alteration,including ones in PTPN11,NF1,NRAS,KRAS and CBL,we identified secondary mutations in genes involved in signal transduction,splicing,Polycomb repressive complex 2(PRC2)and transcription,such as SETBP1,JAK3,JAK2,ASXL1,EZH2,FOXN1 and SRSF2.Using the number of somatic events at diagnosis,patients with one somatic alteration at diagnosis had improved outcomes in comparison to patients who had only one alterations(event-free survival 27.3%±13.4%.vs.74.1%±9.2%,P=0.013).Differences in event-free survival for groups stratified by Ras pathway driver alteration were not statistically significant in our cohort(P= 0.139).A JAK2 missense mutation,a large heterozygous deletion involving JAK3,and homozygous deletion involving RB1 and CDKN2 A were detected in JMML when transforming to AML.After filtering,we reported a novel mutation in RPS19 and a novel mutation in RPL35 A in DBA.The heterozygous mutation,c.383A>C in RPS19 and c.16T>G in the RPL35 A gene were identified and confirmed.We described two mutations,TINF2 c.865C>G and TINF2 c.1241A>G were not reported so far,causing severe clinical phenotype in patients.Conclusion: The number of somatic alterations present at diagnosis,rather than the type of mutations,possessed prognostic relevance.Although previous genomic and biochemical analyses in JMML had emphasized dependence on the Ras pathway for initiation of disease,additional pathways appeared be important for both disease initiation and progression.The present study identified two novel RP gene mutations in Chinese patient with DBA and two novel TINF2 mutations in DC,indicating NGS as an effective and powerful diagnostic tool for rare haematological disorders.