Investigation And Molecular Pathological Mechanism Of A Familial Chronic Myeloproliferative Neoplasm

Objective: Myeloproliferative neoplasms(MPN)are clonal proliferative diseases of bone marrow precursor cells caused by acquired somatic mutations in multipotential hematopoietic stem cells.Classical MPN include Polycythemia vera,Essential thrombocythemia and Primary myelofibrosis.The presence of two or more MPN members in a single family line can be referred to as familial MPN.We identified two patients with MPN with clinical phenotypes of ET and PMF,both positive for CALR mutations,respectively.Next-generation sequencing of the concerned members of this family line was performed,and their raw data were analyzed to find susceptible germline mutations associated with CALR gene mutations,providing a basis for the current pathogenesis of familial MPN.Methods: 1.Family lineage survey: 2 patients with MPN attending the hematology clinic of Sanya Central Hospital were located in the same family,and after informed consent,family members were interviewed for relevant information and family mapping was performed;2.Sample collection: Peripheral blood specimens were collected from 2 patients with pre-existing evidence of MPN with CALR gene mutation and a total of 6 members of their three generations.3.Next-generation sequencing:Extract genomic DNA of each sample,based on Illumina sequencing platform.My Genostics Inc was used for capture library building,and Paired-End method was applied to complete second-generation sequencing of 6 samples with effective sequencing depth of H(100X)and average Q≥80%.Analysis of raw second-generation sequencing data to find germline mutations that were co-existing in 2 patients but not present in healthy members;4.Evaluation: To find the presence of mutated genes that are shared among individual family members,and to evaluate germline mutations with greater association by using SIFT and Polyphen gene prediction tools.Results: 1.The family line involved a total of six members in three generations,three males and three females,one of whom was an ET patient carrying a CALR gene mutation,another was a PMF patient carrying a CALR gene mutation,and another was an anemic,transfusiondependent patient with an unknown diagnosis.All participants signed an informed consent form;2.Based on the raw data of Next-generation sequencing,three susceptible germline mutations were identified as possible pathogenic mutations TDRD9:c.2209C>T(p.R737X),SCN1B:c.744＿749delins ACAAAC(p.S248＿R250delins RQT),NPHS1:c.1802G>C(p.G601A);3.SIFT and Polyphen prediction software indicated that the NPHS1 gene was the most pathogenic.Conclusions: 1.Patients with CALR mutations have a slower disease course;2.ASXL1,CTLA4 may influence the clinical phenotype and prognosis of MPN;3.NPHS1 and SCN1B are present in only two prevalent individuals and may be susceptibility genes for familial MPN.