Associations between a fetal imprinted gene allele score and late pregnancy maternal glucose concentrations.

AIM: We hypothesised that some of the genetic risk for gestational diabetes (GDM) is due to the fetal genome affecting maternal glucose concentrations. Previously, we found associations between fetal IGF2 gene variants and maternal glucose concentrations in late pregnancy. METHODS: In the present study, we tested associations between SNP alleles from 15 fetal imprinted genes and maternal glucose concentrations in late pregnancy in the Cambridge Baby Growth and Wellbeing cohorts (1160 DNA trios). RESULTS: Four fetal SNP alleles with the strongest univariate associations: paternally-transmitted IGF2 rs10770125 (P-value=2×10-4) and INS rs2585 (P-value=7×10-4), and maternally-transmitted KCNQ1(OT1) rs231841 (P-value=1×10-3) and KCNQ1(OT1) rs7929804 (P-value=4×10-3), were used to construct a composite fetal imprinted gene allele score which was associated with maternal glucose concentrations (P-value=4.3×10-6, n=981, r2=2.0%) and GDM prevalence (odds ratio per allele 1.44 (1.15, 1.80), P-value=1×10-3, n=89 cases and 899 controls). Meta-analysis of the associations including data from 1367 Hyperglycaemia and Adverse Pregnancy Outcome Study participants confirmed the paternally-transmitted fetal IGF2/INS SNP associations (rs10770125, P-value=3.2×10-8, rs2585, P-value=3.6×10-5) and the composite fetal imprinted gene allele score association (P-value=1.3×10-8), but not the maternally-transmitted fetal KCNQ1(OT1) associations (rs231841, P-value=0.4; rs7929804, P-value=0.2). CONCLUSION: This study suggests that polymorphic variation in fetal imprinted genes, particularly in the IGF2/INS region, contribute a small but significant part to the risk of raised late pregnancy maternal glucose concentrations.

A cell model for conditional profiling of androgen-receptor-interacting proteins.

Partial androgen insensitivity syndrome (PAIS) is associated with impaired male genital development and can be transmitted through mutations in the androgen receptor (AR). The aim of this study is to develop a cell model suitable for studying the impact AR mutations might have on AR interacting proteins. For this purpose, male genital development relevant mouse cell lines were genetically modified to express a tagged version of wild-type AR, allowing copurification of multiprotein complexes under native conditions followed by mass spectrometry. We report 57 known wild-type AR-interacting proteins identified in cells grown under proliferating and 65 under nonproliferating conditions. Of those, 47 were common to both samples suggesting different AR protein complex components in proliferating and proliferation-inhibited cells from the mouse proximal caput epididymus. These preliminary results now allow future studies to focus on replacing wild-type AR with mutant AR to uncover differences in protein interactions caused by AR mutations involved in PAIS.

Differential gene expression in the striatum of mice with very low expression of the vesicular monoamine transporter type 2 gene.

The vesicular monoamine transporter type 2 (VMAT2) packages pre-synaptic monoamines into vesicles. Previously, we generated mice hypomorphic for the VMAT2 gene (Slc18a2), which results in a approximately 95% reduction in VMAT2 protein, disrupted vesicular storage, severe depletion of striatal dopamine and mice with moderate motor behaviour deficits. Dopamine released from mid-brain dopamine neurons acts on post-synaptic type 1 (D1) and 2 (D2) receptors located on striatal medium spiny neurons to initiate a signalling cascade that leads to altered transcription factor activity, gene expression and neuronal activity. We investigated striatal gene expression changes in VMAT2hypo mice by quantitative real-time PCR and in situ hybridisation. Despite unaltered expression of D1 and D2 dopamine receptors, there were dramatic alterations in striatal mRNAs encoding the neuropeptides substance P, dynorphin, enkephalin and cholecystokinin. The promoters of these genes are regulated by a combination of transcription factors that includes cAMP responsive element binding protein-1 (CREB) and c-Fos. Indeed, the changes in peptide mRNAs were associated with elevated expression of Creb1 and c-Fos. These data indicate that striatal dopamine depletion, as a consequence of deficient vesicular storage in this mouse, triggers a complex program of gene expression, consistent with this mouse being an excellent model of Parkinson's disease.

Mice with very low expression of the vesicular monoamine transporter 2 gene survive into adulthood: potential mouse model for parkinsonism.

We have created a transgenic mouse with a hypomorphic allele of the vesicular monoamine transporter 2 (Vmat2) gene by gene targeting. These mice (KA1) have profound changes in monoamine metabolism and function and survive into adulthood. Specifically, these animals express very low levels of VMAT2, an endogenous protein which sequesters monoamines intracellularly into vesicles, a process that, in addition to being important in normal transmission, may also act to keep intracellular levels of the monoamine neurotransmitters below potentially toxic thresholds. Homozygous mice show large reductions in brain tissue monoamines, motor impairments, enhanced sensitivity to dopamine agonism, and changes in the chemical neuroanatomy of the striatum that are consistent with alterations in the balance of the striatonigral (direct) and striatopallidal (indirect) pathways. The VMAT2-deficient KA1 mice are also more vulnerable to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in terms of nigral dopamine cell death. We suggest that the mice may be of value in examining, long term, the insidious damaging consequences of abnormal intracellular handling of monoamines. On the basis of our current findings, the mice are likely to prove of immediate interest to aspects of the symptomatology of parkinsonism. They may also, however, be of use in probing other aspects of monoaminergic function and dysfunction in the brain, the latter making important contributions to the pathogenesis of schizophrenia and addiction.

Cloning of the mouse organic cation transporter 2 gene, Slc22a2, from an enhancer-trap transgene integration locus.

A novel mouse gene, associated with the enhancer-trap mutation TKZ736, has been cloned and sequenced. It encodes a polyspecific transmembrane transporter with 12 putative transmembrane domains, that shares significant homology with the mouse organic cation transporter 1 (Oct1/Slc22a1) called Lx1. Like Oct1/Slc22a1/Lx1, this gene maps to the proximal part of Chromosome (Chr) 17, but shows a different expression pattern from Oct1/Slc22a1/Lx1. The gene identified here is predominantly expressed in the kidney and ureter, but no expression is detectable in liver. Sequence comparisons suggest that this novel gene most likely represents the mouse homolog of the rat organic cation transporter 2 gene. The genomic DNA flanking the 3' transgene integration site in the enhancer-trap mutation TKZ736 encodes the second exon of the Oct2/Slc22a2 gene.

Structural organization of the human vesicular monoamine transporter type-2 gene and promoter analysis using the jelly fish green fluorescent protein as a reporter.

The genomic structure of a human vesicle monoamine transporter, type-2 (hVMAT2) was determined from two overlapping cosmids, phVMAT2-cos1 and phVMT2-cos2, spanning more than 35 kb. The hVMAT2 open reading frame is encoded by 16 exons, with translation initiation and termination in exon 2 and exon 16, respectively. Several potential binding sites for transcriptional regulatory factors, including a cAMP response element (CRE) were identified in the 5'-upstream region of the gene. A promoter construct using the jellyfish green fluorescent protein (GFP) as reporter has been made and transfected into the human neuroblastoma cell line, SHSY-5Y. The cellular expression of the GFP was readily detected by fluorescence microscopy and cells expressing GFP could be sorted using a fluorescence-activated cell sorter (FACS), allowing the level of GFP expression in transfected SHSY-5Y cells to be quickly and reliably determined.

Structure and expression of a gene encoding a putative GTP-binding protein identified by provirus integration in a transgenic mouse strain.

The Mov-10 mouse strain was derived by infection of preimplantation embryos with the Moloney murine leukemia virus and carries one copy of the provirus in its germ line. Here we show that the provirus has integrated into an evolutionarily conserved gene that can code for a protein of 110 kDa containing the three consensus elements characteristic for GTP-binding proteins. The Mov-10 locus was expressed in a variety of cell types, including embryonal carcinoma and embryonic stem cells. Transcription of the gene was down-regulated about 10-fold when F9 embryonal carcinoma cells are differentiated into parietal endodermlike cells and about 2-fold when they are differentiated into visceral endodermlike cells. High levels of Mov-10 transcripts were also found at different stages of embryonal development and in the testes and thymus of adult animals. Expression was cell cycle controlled, with steady-state RNA levels significantly higher in growth-arrested than in growth-stimulated cells. The results suggest that the Mov-10 locus has an important function in development and/or control of cell proliferation. The provirus was shown to have integrated into intron 1 of the gene without disrupting expression, indicating that integration into intronic sequences of a transcription unit does not necessarily affect transcription. This result together with previous results from the Mov-13 mouse strain suggested that proviruses exert their mutagenic effect only by integration in specific sites, such as cis-regulatory DNA elements.

Retroviral integration sites in transgenic Mov mice frequently map in the vicinity of transcribed DNA regions.

Transcription of cellular sequences flanking proviral insertion sites was studied in several Mov mouse strains, each of which carried one copy of the Moloney murine leukemia virus in its germ line. In three out of five randomly chosen Mov strains, the provirus had integrated in the vicinity of DNA regions transcribed in the embryonal stem cell line CCE and the embryonal carcinoma cell line F9. Assuming that CCE and F9 cells are developmentally equivalent to the early embryonic cells that were infected to establish the Mov strains, our results suggest that retroviruses integrate preferentially into actively transcribed DNA regions.

Retrovirus-induced insertional mutation in Mov13 mice affects collagen I expression in a tissue-specific manner.

In the Mov13 mouse mutant, transcription of the alpha 1 (1) collagen gene is blocked by a retroviral insert in the first intron. We now report that teeth derived from homozygous embryos produce a dentin layer containing normal amounts of collagen 1. In situ hybridization and RNAase protection experiments indicate that the mutant allele is efficiently transcribed in odontoblasts, in contrast to other cell types. Correct splicing of the primary transcript containing the viral sequence results in a functional alpha 1 (1) collagen mRNA. The absence of a mutagenic effect in odontoblasts, as opposed to fibroblasts, suggests that the retroviral insert interferes with tissue-specific transcriptional control of the alpha 1 (1) collagen gene, most likely by inactivating cell-type-specific cis-acting regulatory elements.