Jiahui Xia

Transcriptional regulation of APH-1A and increased γ-secretase cleavage of APP and Notch by HIF-1 and hypoxia

The proteolytic cleavage of Alzheimer β‐amyloid precursor protein (APP) and signaling receptor Notch is mediated by the PS/γ‐secretase complex, which consists of presenilins, nicastrin, APH‐1, and PEN‐2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. Here we characterized the 5′‐flanking region of the human APH‐1A gene and identified a 271‐bp fragment containing the transcription initiation site for the promoter activity. Sequence analysis, mutagenesis, and gel shift studies revealed a binding of AP4 and HIF‐1 to the promoter, which affects the promoter activity. Activation of HIF‐1 by short‐term NiCl2 treatments (a condition of chemical hypoxia) dramatically increased APH‐1A mRNA and protein expression, accompanied by increased secretion of Aβ and decreased APP CTFs formation, indicative of an increase in γ‐secretase activity. NiCl2 treatments had little effect on APP and the other three components of the γ‐secretase complex. The cellular concentration of Notch intracellular domain (NICD) was also increased by the hypoxic treatment. Our results demonstrate that APH‐1A expression and the γ‐secretase mediated Aβ and Notch NICD generation are regulated by HIF‐1, and the specific control of APH‐1A expression may imply physiological functions uniquely assigned to APH‐1A.— Wang, R., Zhang, Y‐W., Zhang, X., Liu, R., Zhang, X., Hong, S., Xia, K., Xia, J., Zhang, Z., Xu, H. Transcriptional regulation of APH‐1A and increased γ‐secretase cleavage of APP and Notch by HIF‐1 and hypoxia. FASEB J. 20, E614–E622 (2006)

Sodium chloride modified silica nanoparticles as a non-viral vector with a high efficiency of DNA transfer into cells.

Development of reliable vectors is a major challenge in gene therapy. Previous gene transfer methods using non-viral vectors, such as liposomes or nanoparticles, have resulted in relatively low levels (35 to approximately 50%) of gene expression. We have developed a silicon nanoparticle (SNAP) system, a novel non-viral vector, for DNA transfer into cells. SNAP was synthesized chemically and modified with sodium chloride or sodium iodide. Electronmicroscopy of SNAP and fluorescence microscopy of fluorescence-labeled SNAP revealed that they were generated uniformly, had diameters of 10-100 nm, and showed a better efficiency (about 70%) of DNA transfection into cells as well as protection of DNA against degradation. The microscopy also demonstrated the adhesion of SNAP with HT1080 cell surface and entry of SNAP into the cells without cytotoxicity. Intravenous and/or intra-abdominal administration of the SNAP to mice revealed the accumulation of SNAP in the cells of the brain, liver, spleen, lung, kidney, intestine, prostate and the testis without any pathological cell changes or mortality, suggesting that they passed through the blood-brain, blood-prostate, and blood-testis barriers. These findings indicate that the SNAP generated has good biological characteristics as a potential promising vector for gene transfer, gene therapy and drug delivery.

Mutation analysis of hereditary multiple exostoses in the Chinese

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2, the two genes responsible for EXT1 and EXT2, respectively, have been cloned. Recently, three other members of the EXT gene family, named the EXT-like genes (EXTL: EXTL1, EXTL2, and EXTL3), have been isolated. EXT1, EXT2, and the three EXTLs are homologous with one another. We have identified the intron-exon boundaries of EXTL1 and EXTL3 and analyzed EXT1, EXT2, EXTL1, and EXTL3, in 36 Chinese families with EXT, to identify underlying disease-related mutations in the Chinese population. Of the 36 families, five and 12 family groups have mutations in EXT1 and EXT2, respectively. No disease-related mutation has been found in either EXTL1 or EXTL2, although one polymorphism has been detected in EXTL1. Of the 15 different mutations (three families share a common mutation in EXT2), 12 are novel. Most of the mutations are either frameshift or nonsense mutations (12/15). These mutations lead directly or indirectly to premature stop codons, and the mutations generate truncated proteins. This finding is consistent with the hypothesis that the development of EXT is mainly attributable to loss of gene function. Missense mutations are rare in our families, but these mutations may reflect some functionally crucial regions of these proteins. EXT1 is the most frequent single cause of EXT in the Caucasian population in Europe and North America. It accounts for about 40% of cases of EXT. Our study of 36 EXT Chinese families has found that EXT1 seems much less common in the Chinese population, although the frequency of the EXT2 mutation is similar in the Caucasian and Chinese populations. Our findings suggest a possibly different genetic spectrum of this disease in different populations.

Molecular analysis of hearing loss associated with enlarged vestibular aqueduct in the mainland Chinese: a unique SLC26A4 mutation spectrum.

AbstractIt has been shown that mutations in the SLC26A4 gene are involved in syndromic deafness characterized by congenital sensorineural hearing impairment and goitre (Pendreds syndrome), as well as in congenital isolated deafness (DFNB4), both of which are associated with enlarged vestibular aqueduct (EVA). The prevalence of SLC26A4 mutations in Pendreds syndrome is clearly established in many ethnic groups, but the data from Mainland Chinese patients with deafness and EVA remain poor. In this report, 15 patients from 13 unrelated Chinese families with deafness and EVA were analyzed for SLC26A4 using direct sequencing. A total of 15 pathogenic mutations were observed in 11 unrelated families, 4 of which were novel. One mutation, IVS7-2A>G, was most common, accounting for 22.3% (5/22) of all the mutant alleles, and H723R was infrequent. To date, a total of 23 mutations have been reported among the Chinese, 13 of which were unique. In conclusion, EVA could be a radiological marker for SLC26A4 analysis among Mainland Chinese hearing-loss patients, and the SLC26A4 mutation spectrum in the Chinese was different from other reported populations.

Transcriptional regulation of PEN-2, a key component of the gamma-secretase complex, by CREB

ABSTRACT Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimers β-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the γ-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APPα and Aβ without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2.

Pregnancy-related complications and adverse pregnancy outcomes in multiple pregnancies resulting from assisted reproductive technology: a meta-analysis of cohort studies

OBJECTIVE To provide an up-to-date comparison of pregnancy-related complications and adverse pregnancy outcomes of multiple pregnancies generated with assisted reproductive technology (ART) vs. spontaneous conception. DESIGN Meta-analysis. SETTING University-affiliated teaching hospital. PATIENT(S) Multiple pregnancies conceived by ART or naturally. INTERVENTION(S) Searches through October 2014 were conducted on PubMed, Google Scholar, Cochrane Libraries, China Biology Medicine disc, Chinese Scientific Journals Fulltext Database, China National Knowledge Infrastructure, and Wanfang Data, to identify studies that met prestated inclusion criteria. Either a fixed- or a random-effects model was used to calculate the overall combined risk estimates. Subgroup analysis was performed to explore potential heterogeneity moderators. MAIN OUTCOME MEASURE(S) Pregnancy-related complications and adverse pregnancy outcomes. RESULT(S) Thirty-nine cohort studies involving 146,008 multiple births were included in the meta-analysis. Multiple pregnancies from ART were associated with a higher risk of premature rupture of membranes (relative risk [RR] = 1.20, 95% confidence interval [CI]: 1.05-1.37; I(2) = 15%); pregnancy-induced hypertension (RR = 1.11, 95% CI: 1.04-1.19; I(2) = 6%); gestational diabetes mellitus (RR = 1.78, 95% CI: 1.25-2.55; I(2) = 42%); preterm birth (RR = 1.08, 95% CI: 1.03-1.14; I(2) = 83%); very preterm birth (RR = 1.18, 95% CI: 1.04-1.34; I(2) = 79%); low birth weight (RR = 1.04, 95% CI: 1.01-1.07; I(2) = 47%); very low birth weight (RR = 1.13, 95% CI: 1.01-1.25; I(2) = 62%); and congenital malformation (RR = 1.11, 95% CI: 1.02-1.22; I(2) = 30%). The relevant heterogeneity moderators have been identified by subgroup analysis. Sensitivity analysis yielded similar results. No evidence of publication bias was observed. CONCLUSION(S) Although the role of potential bias and evidence of heterogeneity should be carefully evaluated, the present study suggests that multiple pregnancies generated via ART, vs. spontaneous conception, are associated with higher risks of pregnancy-related complications and adverse pregnancy outcomes. Further research is needed to determine which aspect of ART poses the most risk and how this risk can be minimized.

A ZRS duplication causes syndactyly type IV with tibial hypoplasia

A ZRS Duplication Causes Syndactyly Type IV With Tibial Hypoplasia Lingqian Wu,* Desheng Liang, Norio Niikawa, Fen Ma, Miao Sun, Qian Pan, Zhigao Long, Zhongmin Zhou, Koh-ichiro Yoshiura, Hua Wang, Daisuke Sato, Gen Nishimura, Heping Dai, Xue Zhang, and Jiahui Xia National Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China Solution Oriented Research of Science and Technology (SORST), Japan Science and Technology Agency (JST), Kawaguchi, Japan Research Institute of Personalized Health Sciences, Health Sciences University of Hokkaido, Hokkaido, Japan McKusick-Zhang Center for Genetic Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Women and Children’s Hospital of Hunan Province, Changsha, Hunan, China Department of Radiology, Tokyo Metropolitan Kiyose Children’s Hospital, Tokyo, Japan

A syndactyly type IV locus maps to 7q36

AbstractSyndactyly occurs as an isolated abnormality or a part of a malformation syndrome. Syndactyly types I, II, III and V have been mapped to chromosomal regions 2q34-q36, 2q31-q32, 6q21-q23.2 and 2q31-q32, respectively, whereas syndactyly type IV (SD4) is extremely rare, and its gene localization has not yet been assigned. The SD4 manifests complete syndactyly of all fingers accompanied with polydactyly, and flexion of the fingers gives the hand a cup-shaped appearance. We performed a linkage and haplotype analysis of a Chinese pedigree with autosomal dominant, non-syndromic SD4 using a set of 406 microsatellite markers. The analysis gave the maximum two-point LOD score of 1.613 at recombination fraction of 0.00 and penetrance of 1.00. Thus, the SD4 locus in the family was likely assigned to a 17.39-cM region at a segment between markers D7S3070 and D7S559 at 7q36, although the LOD score obtained was not high enough to conclude the localization. Analysis of three candidate genes, LMBR1, SHH and ZRS, failed to identify any pathogenic mutations. Our gene mapping may give a clue to identify the putative SD4 gene and provide a better understanding of normal human limb development.

Non‐viral ex vivo transduction of human hepatocyte cells to express factor VIII using a human ribosomal DNA‐targeting vector

Summary.  Background: In gene therapy, one of the most important issues is the choice of the vectors. pHrneo is a human‐derived vector previously constructed by our group, which can target a foreign gene into a human ribosomal DNA (hrDNA) locus. Methods and results: In this study, we inserted an expression cassette of reconstructive hFVIII (hFVIII‐BDDAK39) to pHrneo to construct a targeting vector: pHrneo‐BDDAK39. Through electroporation of pHrneo‐BDDAK39 into HL7702 cells (human hepatocyte), we identified the homologous recombinants using polymerase chain reaction, and tested the expression of hFVIII–BDDAK39 located at the hrDNA locus. The hFVIII‐BDDAK39 was successfully targeted into the hrDNA locus of HL7702 by pHrneo‐BDDAK39, and the efficiency of site‐specific integration was 1.1 × 10−5. The hFVIII‐BDDAK39 at the hrDNA locus of HL7702 was found to be able to express efficiently (4.3 ± 0.9 ng 10−6 cells 24 h−1). Conclusion: It has been indicated that the targeting vector pHrneo‐BDDAK39 can be used in gene therapy for hemophilia A.

A novel fusion suicide gene yeast CDglyTK plays a role in radio-gene therapy of nasopharyngeal carcinoma

To investigate a novel suicide gene for nasopharyngeal carcinoma (NPC) therapy, the yCDglyTK gene was constructed by fusing yeast cytosine deaminase (CD) and herpes simplex type 1 thymidine kinase. The expression of the yCDglyTK gene was detected by RT-PCR and Western blotting, and its bioactivity was demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. An animal study was carried out in which BALB/C nude mice bearing yCDglyTK gene-modified tumors were treated with prodrugs and radiation. Our results revealed that the yCDglyTK gene could be expressed in CNE-2 cells in vitro. In MTT analysis, at the transfection rate of 10%, 66% cells were killed. The synergistic effect of CD and TK showed 91% of yCDglyTK-transfected cells were killed with the treatment of 5-fluorocytosine (5-FC) alone, 60% killed with ganciclovir (GCV) alone, and 75% killed with 5-FC and GCV together. In vivo, the tumor volume in all of the four prodrugs and/or radiation-treated groups were significantly different from that in the PBS-controlled group (P<.01); also yCDglyTK+prodrug+radiation group was different from the other three groups (P<.05). Our findings suggested there was a synergistic antitumor effect when combining suicide gene therapy and radiation, and yCDglyTK has potent antitumor efficacy and may be a candidate suicide gene for cancer therapy.