Genotype-phenotype analysis of a homozygous familial hypercholesterolemia pedigree / 中华儿科杂志

Objective@#To analyze the genetic characteristics of a five generations pedigree with homozygous familial hypercholesterolemia (HoFH).@*Methods@#Prospective study. Twenty family members included a proband diagnosed as familial hyperlipidemia at the cardiology Department of Xi′an Children′s Hospital in October 2018 were research object. Clinical data were collected. Genome DNAs were extracted. Whole exons sequencing was performed on the proband using target capture next generation sequencing. Candidate gene mutation sites identified by bioinformatics were verified by Sanger sequencing in the family members. The genotype-phenotype correlation of the pedigree was analyzed between heterozygous mutation carriers and non-carriers.@*Results@#The proband was a 7-years and 10-month-old boy. He was born with a roundgreen bean size yellow skin protuberance in the skin of the coccyx. Since the age of 3-4 years old, xanthoma-like lesions with a diameter of 0.5-1.5 cm gradually appeared in the skin of bilateral elbow joints, knee joints and Achilles tendon. The height, weight and intellectual development of the child were the same as those of normal children at the same age. No similar xanthoma-like lesion was found in the other family members. The proband′s total cholesterol (TC) reached 18.16-21.24 mmol/L, and his low density lipoproteincholesterol (LDL-C) was 14.08-15.51 mmol/L. Carotid ultrasonography showed diffuse sclerotic plaques in bilateral carotid and vertebral arteries, and color Doppler echocardiography revealed aortic valve thickening and calcification. Gene testing identified that the proband carried a homozygous mutation C. 418G>A (p. E140K) in LDLR gene inherited from his parents who had a consanguineous marriage and carried a heterozygous mutation of LDLR-E140K, respectively.The TC, LDL-C and apolipoproteinB (ApoB) of LDLR-E140K gene heterozygous carriers ((8.40±0.13), (6.79±0.01) and (1.95±0.05) mmol/L, respectively) were significantly higher than those of non-carriers ((4.59±0.28), (3.35±0.39) and (0.86±0.10) mmol/L, t=7.269, 4.595, 6.311, respectively, P<0.05).@*Conclusions@#LDLR-E140K gene homozygous mutation is first reported to be associated with most severe phenotype HoFH. The genotype-phenotype analysis of the pedigree shows that the clinical phenotype of the proband with homozygous mutation is the most serious, and all the heterozygous mutation carriers present with hypercholesterolemia phenotype. The investigation confirms that LDLR-E140K is the pathogenic variation of familial hyperlipidemia.

Study on the anti-tumor efficacy induced by heat shock protein 70-peptide complexes derived from tumor cells / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells.</p><p><b>METHODS</b>Cells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used.</p><p><b>RESULTS</b>HSP70-PC immunization rendered protective effect to both naive tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P < 0.01). CD8+ subset of T lymphocytes in the peripheral blood of immunized mice increased by 12%.</p><p><b>CONCLUSION</b>HSP70-PC induces anti-tumor immunity via activation of cytotoxic T lymphocytes (CTLs), and it possesses strong tumor vaccine effect. Our research adds more evidence to support the clinical use of HSP70-PC to fight human cancers.</p>

Transcriptional activation function of intracellular domain of EPOR and G-CSFR protein in yeast: a comparative study / 解放军医学杂志

Objective To investigate the feasibility of searching for proteins which interact with intracellular domain of hematopoietic growth factor receptors using yeast two-hybrid system. Methods RT-PCR method was performed to amplify the genes of intracellular domains of G-CSF receptor and EPO receptor in NFS-60 and BET-2 cells of mice. The genes were cloned into yeast expression plasmid pGBKT7 vector,and then transformed into yeast AH109. The yeast proteins were isolated and analyzed with Western blotting. Transcriptional activation was analyzed by the ?-galactosidase colony-lift filter assay. Results The intracellular domains of G-CSF receptor and EPO receptor genes were successfully cloned into pGBKT7 vector. The results of Western blotting assay showed that both proteins were expressed in the yeast cells. The ?-galactosidase colony-lift filter assay demonstrated that G-CSF receptor alone had no activity of transcriptional activation,while the EPO receptor alone could activate transcription. Conclusion The findings suggested that intracellular domain of G-CSF receptor could be used as a bait to find interacting proteins using yeast two-hybrid system,while that of the EPO receptor could not. Therefore the system could not be applied to all hematopoietic factor receptor.

Sub-cloning and soluble indution of CILP-MBP recombinant protein by tempera-ture variable / 中国免疫学杂志

Abstract Objective: To construct the Second half of CILP(C2) MBP fusion protein by sub-cloning technology. Methods: Recombinantfusion proteins, which contain the fragments within the C2 region(designated C2F1, C2F2 and C2F3) of the non-porcine nucleotide pyrophos-phohydrolase-homologous region of CILP, were prepared using pMAL-eHis vector. The recombinat genes are induced by different temperatures(22℃,30℃,37℃ ). Results: Expression using pMAL-eHis system can be induced chemically by adding IPTG. 37℃ temperature prmotes in-soluble inclusion-body formation,but 22℃ temperature can not induce the enough expression of recombinant protein. Onl 30℃ temperaturecan induce enough amount of soluble recombinant protein. The characers of fusion proteins that they carried 6 straight histidines, (His)6, at tbeC-terminus of multiple cloning sites for affinity purification were assessed by sodium dodecy1 sulfate-polyacylamide gel electrophoresis(SDS-PAGE) and Western blot. Nucleotide sequences of the insertion genes were confirmed by dideoxy sequencing. Conclusion: C2F1, C2F2,C2F3-MBP fnsion proteins were constructed successfully.These recombinant proteins may provide important roles in the future study on CILP.