Molecular Diagnosis And Gene Therapy Of Hereditary Hemorrhagic Diseases

Objectives:1.This study aims to analyze the molecular pathology of bleeding disorders and establish a platform for the molecular diagnosis and differential diagnosis of intractable hereditary hemorrhagic diseases via next-generation sequencing,DNA microarray,and Sanger sequencing techniques.Two cases of intractable hemorrhagic diseases are analyzed in this study.Through the study of a case of 22q11 microdeletion syndrome,we try to explore new candidate causative genes and their molecular pathology to deepen our understanding of this syndrome.Another case of hemoglobin Hornchurch complicated with thrombocytopenia reveals the characteristics of hemoglobin Hornchurch.2.This study aims to establish models for the study of hereditary bleeding disorders by using the CRISPR/Cas9 technology and conduct preliminary explorations into gene therapy,which may provide new strategies for gene therapy of single-gene diseases.Specifically,we try to establish a cell line harboring the alpha-1-antitrypsin Pittsburgh mutation by CRISPR/Cas9 to lay the foundation for gene therapy research.Methods:1.Specimens were collected from bleeding patients without clear diagnosis from the Department of Hematology of the First Affiliated Hospital of Soochow University during 2015-2017.Gene sequencing was used to diagnose these patients and confirmed patients' clinical and laboratory features were summarized.2.A cell line harboring the alpha-1-antitrypsin Pittsburgh mutation was established by CRISPR/Cas9 technology.Real-time quantitative PCR(q-PCR)and Western Blot(WB)were performed to detect gene expression at m RNA and protein levels.Results:1.Exploration of new candidate causative genes for 22q11 microdeletion syndromeMicroarray analysis revealed a microdeletion of 125 kb(chromosomes 22:24276973-24402263)in the case of 22q11 microdeletion syndrome,including 8 genes: GSTT2 B,GSTT2,DDTL,DDT,GSTTP1,LOC391322,GSTT1 and GSTTP2.These genes are not involved in known causative genes for this syndrome.They may regulate the development of the third and fourth pharyngeal arches,or participate in the regulation of known causative genes for 22q11 DS abnormalities.2.Clinical and laboratory characteristics of a hemoglobin Hornchurch patientBy sequencing the HBB gene,we diagnosed a patient with hemoglobin Hornchurch and summarized his clinical and laboratory characteristics.In contrast to several cases that have been reported,this case showed positive for the isopropanol precipitation test,revealing that the relationship between structural abnormalities and functional properties of Hb Hornchurch requires further research.On the other hand,the second case of Hb Hornchurch in the Chinese population suggests that there may be more Hb variants or carriers in the Chinese population.3.Establishment of a cell line harboring the alpha-1-antitrypsin Pittsburgh mutationWe successfully established a cell line barboring the alpha-1-antitrypsin Pittsburgh mutation using the CRISPR/Cas9 technology.Conclusion:1.In this study,genetic diagnosis was performed for bleeding cases without confirmed diagnosis.Several rare hemorrhagic diseases were diagnosed and the corresponding clinical features and laboratory characteristics were analyzed.At the same time,the important role of gene sequencing in the diagnosis of hereditary hemorrhagic diseases was revealed..2.Eight possible new causative genes of 22q11 microdeletion syndrome were discovered by microarray analysis in a 22q11 DS patient: GSTT2 B,GSTT2,DDTL,DDT,GSTTP1,LOC391322,GSTT1 and GSTTP2.Positive result for the isopropanol precipitation test was found in a patient with hemoglobin Hornchurch,suggesting that Hb Hornchurch is not stable to some extent,and that there may be more variants of Hb or its carriers in the Chinese population.3.The cell line barboring the alpha-1-antitrypsin Pittsburgh mutation was successfully established by the CRISPR/Cas9 technology,which laid the foundation for subsequent gene therapy research.