Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia(FA).However,mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes.Herein,we intr...Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia(FA).However,mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes.Herein,we introduced two novel diagnostic methods,including single-cell sequencing and capillary nano-immunoassay.One FA case with FANCM c.4931G>A p.R1644Q and FANCD1 c.6325G>A p.V2109I was studied.The DNA of 28 cells was amplified and eight types of cells were observed after Sanger sequencing.There were two homozygous mutations(FANCM/FANCD1).Furthermore,the capillary nano-immunoassay was conducted to analyze the expression profile of FA-associated proteins.Abnormal FANCM and FANCD1 expressions simultaneously existed.This case was thus diagnosed as FA-D1/FA-M dual subtype.Compared with mixed cell sequencing,single-cell sequencing data shows more accuracy for the FA subtype evaluation,while the capillary nano-immunoassay is a good method to detect the expression profile of abnormal or modified FA protein.展开更多
Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration,while preventing cell transformation after damage.Loss of PUMA dramatically incre...Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration,while preventing cell transformation after damage.Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation(IR),while without promoting tumorigenesis in the long-term survivors.This finding suggests that PUMA(p53 upregulated modulator of apoptosis)may have a function other than regulates apoptosis.Here,we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells(PSCs)and immortalized hematopoietic progenitor cells(HPCs)after IR.We found that PUMA-deficient PSCs and HPCs exhibited a significant higher doublestrand break(DSB)DNA repair activity via Rad51-mediated homologous recombination(HR).This is because PUMA can be associated with early mitotic Inhibitor 1(EMI1)and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation,thereby inhibiting Rad51 nuclear translocation and HR DNA repair.Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.展开更多
基金This work was partially supported by grants from the National Natural Science Foundation of China(81670112 and 31571380)CAMS Innovation Fund for Medical Sciences(CIFMS,2016-I2M-1-002)The National Key Research and Development Program of China(2016YFC0901503).
文摘Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia(FA).However,mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes.Herein,we introduced two novel diagnostic methods,including single-cell sequencing and capillary nano-immunoassay.One FA case with FANCM c.4931G>A p.R1644Q and FANCD1 c.6325G>A p.V2109I was studied.The DNA of 28 cells was amplified and eight types of cells were observed after Sanger sequencing.There were two homozygous mutations(FANCM/FANCD1).Furthermore,the capillary nano-immunoassay was conducted to analyze the expression profile of FA-associated proteins.Abnormal FANCM and FANCD1 expressions simultaneously existed.This case was thus diagnosed as FA-D1/FA-M dual subtype.Compared with mixed cell sequencing,single-cell sequencing data shows more accuracy for the FA subtype evaluation,while the capillary nano-immunoassay is a good method to detect the expression profile of abnormal or modified FA protein.
基金This work was supported by the grants from the Ministry of Science and Technology of China(2016YFA0100600)the National Natural Science Foundation of China(81730006,81890990,81874078,82072896,81972341,and 81772663)+2 种基金CAMS Initiative for Innovative Medicine(2016-I2M-1-017,2019-I2M-1-006)Shanghai Municipal Science and Technology Commission(19JC1413500)Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant(No.20161310).
文摘Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration,while preventing cell transformation after damage.Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation(IR),while without promoting tumorigenesis in the long-term survivors.This finding suggests that PUMA(p53 upregulated modulator of apoptosis)may have a function other than regulates apoptosis.Here,we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells(PSCs)and immortalized hematopoietic progenitor cells(HPCs)after IR.We found that PUMA-deficient PSCs and HPCs exhibited a significant higher doublestrand break(DSB)DNA repair activity via Rad51-mediated homologous recombination(HR).This is because PUMA can be associated with early mitotic Inhibitor 1(EMI1)and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation,thereby inhibiting Rad51 nuclear translocation and HR DNA repair.Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.