Diseases and pathogenesis of ATP6V1B2 gene mutations / 国际儿科学杂志

Mutations of ATP6V1B2(ATPase H + transporting V1 subunit B2)gene may cause dominant hereditary deafness and onychodystrophy(DDOD, MIM124480)syndrome and Zimmermann-Laband syndrome 2(ZLS2, MIM616455). It is demonstrated that patients with DDOD syndrome have learning or memory problems as well as congenital sensorineural hearing loss and nail aplasia or hypoplasia.ZLS2 is mainly characterized by gingival hypertrophy, hypo/aplastic nails and intellectual disability.Individuals are reported to have intellectual disability, epilepsy and milder ZLS2-type characteristics if they carry the variants clustering towards the middle of the ATP6V1B2 protein.ATP6V1B2 gene encodes B2 subunit of the multimeric vacuolar H + ATPase(V-ATPase), which is a proton pump responsible for controlling the intracellular and extracellular pH of cells and organelles.Research on Atp6v1b2 c. 1516C> T knockin mice showed that homozygous mice displayed obvious cognitive defects and impaired hippocampal CA1 region.A weak interaction between the E and B2 subunits might be the molecular mechanism underlyingV-ATPases dysfunction.It is of great significance to analyze and compare the phenotype caused by pathogenic mutations of ATP6V1B2 gene and to carry out functional research.

Two novel pathogenic mutations of GAN gene identified in a patient with giant axonal neuropathy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the disease-causing mutations in a patient suspected for giant axonal neuropathy(GAN).</p><p><b>METHODS</b>Target sequence capture sequencing was used to screen potential mutations in genomic DNA extracted from peripheral blood sample of the patient. Sanger sequencing was applied to confirm the detected mutation. The mutation was verified among 400 GAN alleles from 200 healthy individuals by Sanger sequencing. The function of the mutations was predicted by bioinformatics analysis.</p><p><b>RESULTS</b>The patient was identified as a compound heterozygote carrying two novel pathogenic GAN mutations, i.e., c.778G>T (p.Glu260Ter) and c.277G>A (p.Gly93Arg). Sanger sequencing confirmed that the c.778G>T (p.Glu260Ter) mutation was inherited from his father, while c.277G>A (p.Gly93Arg) was inherited from his mother. The same mutations was not found in the 200 healthy individuals. Bioinformatics analysis predicted that the two mutations probably caused functional abnormality of gigaxonin.</p><p><b>CONCLUSION</b>Two novel GAN mutations were detected in a patient with GAN. Both mutations are pathogenic and can cause abnormalities of gigaxonin structure and function, leading to pathogenesis of GAN. The results may also offer valuable information for similar diseases.</p>

Isolation and gene modification of amniotic fluid derived progenitor cells / 生物工程学报

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.

An Expedient Reliable Double Fluorescent Reporter System for φC31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for φC31 integrase was developed in NIH3T3 cells. The reporter plasmid coding green fluorescent protein (GFP) coupled with red fluorescent protein (RFP) was eo-transfected with the plasmid coding φC31 integrase, to show the activity of integrase in the cells. Fluorescence activated cell sorter (FACS) was used to measure the proportion of the cells containing red and green fluorescence. The increment of green cells was positively related to the increase in the transfection with plasmid coding φC31 integrase. Approximately 90% of green cells were observed under a ratio of [plasmid-φC31-integrase]/[reporter plasmid] at 10 : 1. This suggests that the φC31 integrase reporter system provides a probe for the function of φC31 integrase in cells.

An Expedient Reliable Double Fluorescent Reporter System for ?C31 Integrase Function Evaluation / 生物化学与生物物理进展

A reporter system for ?C31 integrase was developed in NIH3T3 cells.The reporter plasmid coding green fluorescent protein(GFP) coupled with red fluorescent protein(RFP) was co-transfected with the plasmid coding ?C31 integrase, to show the activity of integrase in the cells.Fluorescence activated cell sorter(FACS) was used to measure the proportion of the cells containing red and green fluorescence.The increment of green cells was positively related to the increase in the transfection with plasmid coding ?C31 integrase.Approximately 90% of green cells were observed under a ratio of plasmid-?C31-integrase/reporter plasmid at 10∶1.This suggests that the ?C31 integrase reporter system provides a probe for the function of ?C31 integrase in cells.

Molecular identification of human/goat xenogeneic model / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the human hematopoietic stem cells from the human/goat xenogeneic model with molecular techniques.</p><p><b>METHODS</b>DNA and total RNA were extracted from 11 transplanted goat peripheral blood cells. Human CD(34), GPA and SRY genes were amplified with PCR in these samples, and CD(34), GPA mRNA transcripts were detected using RT-PCR in 5 and 6 goat peripheral blood cells, respectively. Southern blot analysis was performed in 8 goat DNAs to detect the human specific alpha-satellite sequence. Meanwhile FISH was also performed to detect the human cells in goat blood with a probe of human Y chromosome.</p><p><b>RESULTS</b>Human CD(34) and GPA genes could be detected with PCR in all the 11 goats, and SRY gene did in 5 goats transplanted with hematopoietic stem cells derived from male human babies. Southern blot showed that human specific alpha-satellite sequence was present in 8 goats. By RT-PCR, human CD(34) mRNA was detected in 5 experimental goats, GPA mRNA was found in the other 6 experimental goats and FISH assay showed that some peripheral blood cells of the human/goat xenogeneic model were positive.</p><p><b>CONCLUSION</b>Existence of human cells in the recipient goats was identified by molecular detection, which was feasible for the examination of human/goat xenogeneic models.</p>