A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.

Homozygous mutation Gly542Ser in one pedigree with congenital factor Ⅻ deficiency / 中华检验医学杂志

Objective To analyze the mutations of F12 gene in one pedigree with congenital factor FⅫ(FⅫ)deficiency, and investigate the molecular mechanisms of FⅫ deficiency.Methods Pedigree investigation.In February 2015,a patient with hereditary FⅫdeficiency was admitted to the Third Clinical College of Wenzhou Medical University.Activated partial thromboplastin time(APTT), prothrombin time (PT),FⅫactivity(FⅫ:C),FⅫ antigen(FⅫ:Ag)and other coagulant parameters were tested in the proband and his family members.5′and 3′UTR, all exons and their exon-intron boundaries of F12 gene were analyzed by direct sequencing.The detected mutations were confirmed by reverse sequencing.The conserved amino acids were analyzed by ClustalX-2.1-win software, and four bioinformatics softwares (PolyPhen-2,PROVEAN,SIFT and MutationTaster)were also used to analyze the effect of mutations on protein function.Results The proband and her younger brother showed a markedly prolonged APTT which were 116.4 s and 101.3 s, while her father had slightly prolonged APTT, and other family members were normal.The FⅫ:C and FⅫ:Ag of family members were also decreased(the proband,2.0% and 1.0%;her younger brother,2.0% and 1.0%; her father,18.0% and 13.0%).The phenotype of all members was consistent with cross-reactive material(CRM)negative.Nucleotide sequencing analysis showed that the proband and her younger brother had missense mutations in the F 12 gene, including one homozygous mutation c.1681G>A(p.Gly542Ser)and a commonly reported single nucleotide polymorphism site within the promoter region of the F12 gene(46T/T).Sequencing results from the proband's parents and son demonstrated them as carriers of a heterozygous missense mutation.The proband's husband was normal and with 46C/C in the promoter region.The ClustalX-2.1-win results indicated that the Gly542 was highly conserved among the homologousspecies.The predicting outcomes of the four bioinformatics softwares were the same,the PolyPhen-2(score 1.000)and PROVEAN(score -4.975)both declared p.Gly542Ser was a harmful mutation.The SIFT(score 0.00)and the MutationTaster(score 0.999)manifested the mutation could affect the protein funtion.Conclusions c.1681G>A(p.Gly542Ser)in exon 14 and 46T/T were related with the significant decrease of the FⅫlevel of this pedigree of hereditary FⅫ deficiency.

Features of radionuclide myocardial perfusion imaging in patients with homozygous familial hypercholesterolemia / 中华核医学与分子影像杂志

Objective To evaluate the features of myocardial perfusion imaging (MPI) in patients with homozygous familial hypercholesterolemia (HoFH) and its influence factors.Methods Forty-two consecutive HoFH patients (21 males,21 females;average age:(14.8±8.4) years) were retrospectively enrolled in this study from June 2010 to November 2016.Diagnosis was proved by clinical and chromosome tests,and all patients underwent ATP stress and rest 99Tcm-methoxyisobutylisonitrile (MIBI) SPECT MPI with a two-day protocol.Summed stress score (SSS) and summed rest score (SRS) were acquired,and summed difference score (SDS;SSS-SRS) was calculated.Relations between SSS,SRS,SDS and age,lipid profile were analyzed.Two-sample t test,x2 test,multiple linear regression analysis and multivariate logistic regression were used to analyze the data.Results There were 24 patients with positive MPI results (SSS≥1),and females (76.2％,16/21) showed more positive MPI results than males (38.1％,8/21;x2=6.22,P＜0.05).Eighteen patients had negative MPI results.There were 6,8,10 patients with MPI positive results in ＜ 10 years group (n =14),10-18 years group (n =14) and ≥ 19 years group,respectively (x2=2.33,P＞0.05).Positive electrocardiograph (ECG) in ATP stress test was observed in 9 females (42.9％,9/21) and 3 males (14.3％,3/21;x2 =4.20,P＜0.05).Sixty-three (8.8％,63/714) abnormal myocardial perfusion segments (SSS≥ 1) were found,which was mainly (60.3％,38/63) distributed in myocardial regions supplied by left anterior descending branch (LAD).SSS was positively correlated with age and high density lipoprotein cholesterol (HDLC).SRS,SDS were positively correlated with HDLC and age respectively.Multivariate logistic regression analysis indicated that the female was the only independent risk factor to predict positive MPI (odds ratio=5.2,95％ CI:1.363-19.774).Conclusions In HoFH patients,abnormal myocardial perfusion had a rising trend with age increasing.Female patients are more likely to have abnormal MPI.Abnormal myocardial perfusion segments are mainly located in myocardial regions supplied by LAD.Age and gender are influence factors of abnormal MPI in HoFH patients.

A novel homozygous mutation Leu519Arg in one pedigree with congenital factor XII deficiency / 中华检验医学杂志

To analyze the mutations of F12 genein one pedigree with congenital factor FXII (FXII) deficiency , and investigatethe molecular mechanisms of FXII deficiency . Methods Activated partial thromboplastin time(APTT),Prothrombin time(PT), FXII activity(FXII:C), FXII antigen(FXII:Ag) and other coagulant parameters were tested in the proband and his family members .5'and 3'UTR,all exons and their exon-intron boundaries of F12 gene were analyzed by direct sequencing .The detected mutations were confirmed by reverse sequencing .100 healthy persons were as normal controls .Results The proband showed a markedly prolonged APTT (106.4s), the FXII:C and FXII:Ag were 2.0% and 1.0%, respectively .Hissecond daughter and granddaughter had slightly prolonged APTT , and other family members are normal.The FXII:C and FXII:Ag of family members were also decreased ( his son, 23.0% and 21. 0%;his elder daughter , 23.0%and 23.0%;his second daughter ,24.0%and 23.0%;hisgranddaughter , 23.0%and 23.0%).The phenotype of all members is consistent with cross -reactive material negative . Nucleotide sequencing analysis showed that the proband had missense mutations in the F 12 gene, including one homozygous mutationc.1556T >G ( p.Leu519Arg) and a commonly reported single nucleotide polymorphism site within the promoter region of the F 12 gene (46T/T) .Sequencing results from the proband 'children demonstrate them as carriers of a heterozygous missense mutation .The proband 's wife is normal and with 46C/C in the promoter region .Conclusion The c.1556T>G in exon 13 is a novel mutation .This mutation affects FXIIcatalytic function , associated with a reduced level of FXII .

Breeding and genotype identification of NLRP3 gene knock-out mice / 第二军医大学学报

Objective: To breed and identify NLRP3 gene knock-out mice. Methods: The NLRP3 gene knock-out heterozygote mice were bred alone and copulated. The offsprings were to have three genotypes: wild genotype, heterozygote genotype and homozygote genotype. Genomic DNA was obtained from each pups and were subjected to PCR and T7 endonuclease 1 to identify the genotype. The homozygote mice were mated with the opposite sex heterozygote mice to obtain more homozygote pups. Results: Breeding and reproducing were both successful, and we obtained heterozygote genotype and homozygote genotype mice with NLRP3 gene knock-out. Conclusion: Correct methods of breeding, reproducing and identifying can effectively obtain NLRP3 gene knock-out mice from heterozygote mice.

Cardiac Surgery in Homozygous Familial Hypercholesterolemia / 日本心臓血管外科学会雑誌

Homozygous familial hypercholesterolemia is a rare metabolic disorder with characteristic clinical presentations, such as tendon xanthomas, hypercholesterolemia, and significant cardiovascular disease including premature coronary artery disease. We describe a case of a 56-year-old woman with homozygous familial hypercholesterolemia. She had been treated with low-density lipoprotein apheresis for 23 years. Preoperative echocardiography and coronary angiography showed severe aortic valve stenosis and right coronary artery stenosis. Aortic valve replacement with patch enlargement of the aortic valve annulus, and coronary artery bypass grafting were successfully performed. She was discharged uneventfully.

The influence of human leucocyte antigen-Ⅰ polymorphisms on plasma viral load in human immunodeficiency virus infected male homosexual population in Beijing / 中华传染病杂志

Objective To analyze the influence of the polymorphisms of human leucocyte antigen (HLA)-Ⅰ molecule and the effects on plasma viral load of human immunodeficiency virus (HIV) infected male homosexual population in Beijing.Methods The HLA-A,HLA-B,HLA-C allele were typed by sequence specific primer-polymerase chain reaction (SSP-PCR),and viral load was detected in 157 chronic HIV infected persons.Normally distributed measurement data were analyzed by one-way or multi-way analysis of variance,while data of abnormal distributions were analyzed by Mann-Whitney U test.Results Among 157 chronic HIV infected persons,the number of Bw4 motifs on HLA-B loci was associated with a lower level of viral load (F=3.01,P=0.045).In these HIV infected persons,the viral load in HLA-B carrying Bw4/4 homozygote was (4.19±0.76) lg IU/mL,in HLA-B carrying Bw6/6 homozygote was (4.63±0.74) lg IU/mL (t=2.27,P=0.010).The viral load of those who carried three,one or none Bw4 motifs on HLA-A and HLA-B loci were (3.92± 0.97),(4.54±0.88) and (4.60±0.72) lg IU/mL,respectively (three vs none:t=2.53,P=0.015; three vs one:t=2.11,P=0.039).HIV infected persons who carried homozygote on any loci of HLA-A,HLA-B,HLA-C had comparable levels of plasma viral load to those who carried heterozygote on HLA-A,HLA-B,HLA-C loci.Among the persons who carried heterozygote on HLA-A,HLA B,HLA-C loci,Bw4/4 homozygote on HLA-B had lower levels of viral load than Bw6/6 homozygote on HLA-B (median:4.09 lg IU/mL vs 4.55 lg IU/mL,U=210.50,P=0.041).HIV infected persons who carried A30/B13/C06 or A33/B58/C03 haplotype had comparable levels of plasma viral load to those without A30/B13/C06 or A33/B58/C03 haplotype (t=0.40,P=0.69; t=0.68,P=0.49,respectively).Conclusions Bw4/4 homozygote on HLA-B loci is associated with lower HIV viral load.Furthermore,the plasma viral load of HIV infected persons carrying heterozygote on HLA-A,HLA-B,HLA-C loci could be influenced by the Bw4/4 homozygote on HLA-B locus,with a lower viral load.

Breeding of homozygous mice of Treg-Th17 fluorescence double labeling and their preliminary use in endotoxemia study / 中华创伤杂志

Objective To breed homozygous mice of Treg-Th17 fluorescence double labeling and to investigate the role of Treg-Th17 balance in endotoxemia. Methods Mice of Treg and Th17 fluorescence single labeling were brought from Harvard Medical School,USA and were mated and bred based on the genetic rules.The genomic DNA was extracted from the tails of second generation weaning mice for genotyping by polymerase chain reaction (PCR).Thereafter,the homozygous mice of fluorescence double labeling were selected to have successive inbreeding for three generations and their genotypes and growth status were measured.The endotoxemia model in vivo was duplicated and changes of Treg-Th17 balance were detected by flow cytometry analysis at 24 h and 48 h after endotoxemia. Results Among the seven second generation mice in the first batch,only three (two males and one female) were the wanted homozygotes.Meanwhile,the genotypes of their offsprings for the next three generations were all Foxp3 RFPKI-homo IL-17A GFPKI with normal growing status.As important component of endotoxin,lipopolysaccharide (LPS)could induce significant signal expressions of red fluorescence protein (RFP) and green fluorescence protein (GFP).The ratio of CD8a + Foxp3 +,CD8a + IL-17A +,CD4 + Foxp3 + and CD4 + IL-17A + in the lymphocytes went up significantly in the group of 24 hours of LPS treatment,as compared with the control group (P ＜ 0.01 or P ＜ 0.05 ),while the difference between the control group and the group of 48hours of LPS treatment had no statistical significance (P ＞ 0.05). Conclusion Foxp3 RFPKI-homoIL-17A GFPKI mice have identical genotypes and stable genetic characteristics and is a model animal and novel research platform that can provide real-time monitoring of the changes of Treg-Th17 balance in vivo.The role of T reg-Th17 balance works significantly at 48 hous after the subject is exposed to LPS stimulation,and the roles of CD8a + Foxp3 and CD8a + IL-17A + in Treg-Th17 balance require further investigation.

Gene mutations in low-density lipoprotein receptor in one Chinese padigree with homozygous familial hypercholesterolemia / 中华检验医学杂志

Objective To investigate low density lipoprotein receptor (LDLR)gene mutation in familial hypercholesterolemia (FH) patients. Methods The proband was given clinical diagnosis of homozygous FH based on marked features and blood lipid tests results. After apoB100R3500Q mutation was excluded, the promoter region and all of the 18 exons of LDLR gene were amplified by touch-downpolymerase chain reaction (PCR). The PCR products were analyzed by single-strand conformationalpolymorphism (SSCP). The PCR products with abnormal single strands were sequenced directly. Thesecondary structures of the mutational and wild type proteins were analyzed and compared byANTHEPROT5.0, and then the tertiary structures of the mutant and wild type LDLR were predicted atSWISS MODEL homepage online. Results A homozygous mutation A606T at exon 13 of the patients wasfound by SSCP and confirmed by DNA sequencing. GOR Ⅰ method in ANTHEPROT5.0 indicates that therandom coils and turns would replace some helixes at the mutation site. The online prediction from theSWISS MODEL homepage indicates the backbone structure of the mutant LDLR has no difference from thewild type one. Conclusion The results suggest the A606T mutation of LDLR gene is the cause of the FH inthis pedigree.

Applications of RHD zygosity test through polymerase chain reaction for prediction of fetus Rh D-positive phenotype / 中华妇产科杂志

Objective To explore a molecular method for the determination of RHD zygosity. Methods Two pairs of primers were designed specific for downstream rhesus box and hybrid rhesus box according to the sequences in GenBank. Together with a pair of internal control primer, a dual-tube polymerase chain reaction (PCR) method was established for determination of the RHD zygostity. Thirty Rh D-positive, D-negative and weak D phenotype samples were evaluated by taking a recent restriction fragment length polymorphism (RFLP) method as reference. Results The results of the dual-tube PCR and RFLP are identical in 29 samples, which are also in concordance with the Rh phenotype, respectively. In one weak D sample, however, both methods tested as RHD -/RHD -, which showed that there was one RHD - haplotype in this individual, but a false negative result in testing downstream rhesus box. Conclusions The dual-tube PCR is a less-labored method for RHD zygosity detection. It can be used clinically in prenatal test to determine RHD zygosity of pregnant women′s partner for prediction of fetus RhD phenotype.

Prenatal Gene Diagnosis for High Risk Infant of Thalassemia / 中华围产医学杂志

Objective To evaluate the value of prenatal gene diagnosis for thalassemia. Methods 128 fetuses suspected with thalassemia were performed amniocentesis or cordocentesis for gene diagnosis. Results No severe complications occurred during all procedures. 32 fetuses had normal genotype. 38 were heterozygous and 27 were homozygous of ?- thalassemia; 18 were heterozygous, 4 were homozygous and 9 were double heterozygous of ?-thalassemia. The types and frequencis of ?-thalassemia mutation were CD 41-42(47.5%), IVS-Ⅱ-654(42.5%), 17(A-T)(7.5%) and -28 (A-G)(2.5%) in turn. Pregnancies of 40 fetuses with severe thalassemia were terminated in time. Conclusions The screening and prenatal diagnosis of high risk fetus for thalassemia is safe, effective and accurate. It should be used as an obstetrical routine examination at the region with high thalassemia occurrence.

A Case of 17a-Hydroxylase Deficiency in 17-Year-Old Girl / 대한내분비학회지

The single enzyme P-450c17 hydroxylase catalyzes the 17a-hydroxylation of both pregnenolone and progesterone and the side-chain cleavage of 17a-hydroxypregnenolone and 17a-hydroxypro- gesterone to dehydroepiandrosterone and androstenedione. This enzyme is located in the endoplasmic reticulum and consists of a P-450c17 and a specific flavoprotein NADPH-cytochrome P-450 reductase. The clinical picture and hormonal pattern in 17a-hydroxylase deficiency have been consistent in both genotypic sexes with hypergonadotropic hypogonadism in whom the virtual absence of gonadal steroids results in a female phenotype with primary amenorrhea and pseudohermaphro- ditism in the male and underdeveloped secondary sex characteristics and hypermineralocorticoidism with hypertension, hypokalemia, suppressed renin-angiotensin system and extremely reduced aldo-sterone production. A 17-year-old girl visited endocrine clinic because of amenorrhea, absence of pubic and axillary hair, and hypertension. she had elevated levels of serum corticosterone, deoxycorticosterone(DOC), 18-hydroxycorticosterone(18-OHB). Stumulation with ACTH effected minimal increase in the elevated steroids and the ACTH-stimulated 18-OHB to aldosterone ratio was more than 280. These hormonal patterns appear to be homozygote in 17a-hydroxylase deficiency.