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1.  Multiple congenital melanocytic naevi and neurocutaneous melanosis are caused by post-zygotic mutations in codon 61 of NRAS 
Congenital melanocytic naevi (CMN) can be associated with neurological abnormalities and increased risk of melanoma. Mutations in NRAS, BRAF and Tp53 have been described in individual CMN samples, however, their role in the pathogenesis of multiple CMN within the same subject and development of associated features has not been clear. We hypothesised that a single post-zygotic mutation in NRAS could be responsible for multiple CMN in the same individual, as well as for melanocytic and non-melanocytic central nervous system (CNS) lesions. Fifty-five samples from 15 patients with multiple CMN were sequenced after site-directed mutagenesis and enzymatic digestion of the wild-type allele. Oncogenic missense mutations in codon 61 of NRAS were found in affected neurological and cutaneous tissues of 12/15 patients, but absent from unaffected tissues and blood, consistent with NRAS mutation mosaicism. In ten patients the mutation was consistently c.181C>A, p.Q61K, and in two c.182A>G, p.Q61R. All 11 non-melanocytic and melanocytic CNS samples from five patients were mutation positive, despite NRAS rarely reported as mutated in CNS tumours. Loss of heterozygosity was associated with onset of melanoma in two cases, implying a multi-step progression to malignancy. These results suggest that single post-zygotic NRAS mutations are responsible for multiple CMN and associated neurological lesions in the majority of cases.
doi:10.1038/jid.2013.70
PMCID: PMC3678977  PMID: 23392294
2.  Paternally Expressed, Imprinted Insulin-Like Growth Factor-2 in Chorionic Villi Correlates Significantly with Birth Weight 
PLoS ONE  2014;9(1):e85454.
Context
Fetal growth involves highly complex molecular pathways. IGF2 is a key paternally expressed growth hormone that is critical for in utero growth in mice. Its role in human fetal growth has remained ambiguous, as it has only been studied in term tissues. Conversely the maternally expressed growth suppressor, PHLDA2, has a significant negative correlation between its term placental expression and birth weight.
Objective
The aim of this study is to address the role in early gestation of expression of IGF1, IGF2, their receptors IGF1R and IGF2R, and PHLDA2 on term birth weight.
Design
Real-time quantitative PCR was used to investigate mRNA expression of IGF1, IGF2, IGF1R, IGF2R and PHLDA2 in chorionic villus samples (CVS) (n = 260) collected at 11–13 weeks' gestation. Expression was correlated with term birth weight using statistical package R including correction for several confounding factors.
Results
Transcript levels of IGF2 and IGF2R revealed a significant positive correlation with birth weight (0.009 and 0.04, respectively). No effect was observed for IGF1, IGF1R or PHLDA2 and birth weight. Critically, small for gestational age (SGA) neonates had significantly lower IGF2 levels than appropriate for gestational age neonates (p = 3·6×10−7).
Interpretation
Our findings show that IGF2 mRNA levels at 12 weeks gestation could provide a useful predictor of future fetal growth to term, potentially predicting SGA babies. SGA babies are known to be at a higher risk for type 2 diabetes. This research reveals an imprinted, parentally driven rheostat for in utero growth.
doi:10.1371/journal.pone.0085454
PMCID: PMC3893199  PMID: 24454871
3.  Fat dads must not be blamed for their children's health problems 
BMC Medicine  2013;11:30.
The relationship between the parental genomes in terms of the future growth and development of their offspring is not critical. For the majority of the genome the tissue-specific gene expression and epigenetic status is shared between the parents equally, with both alleles contributing without parental bias. For a very small number of genes the rules change and control of expression is restricted to a specific, parentally derived allele, a phenomenon known as genomic imprinting. The insulin-like growth factor 2 (Igf2/IGF2) is a robustly imprinted gene, important for fetal growth in both mice and humans. In utero IGF2 exhibits paternal expression, which is controlled by several mechanisms, including the maternally expressing untranslated H19 gene. In the study by Soubry et al., a correlation is drawn between the IGF2 methylation status in fetal cord blood leucocytes, and the obesity status of the father from whom the active IGF2 allele is derived through his sperm. These data imply that paternal obesity affects the normal IGF2 methylation in the sperm and this in turn alters the expression of IGF2 in the baby.
doi:10.1186/1741-7015-11-30
PMCID: PMC3584737  PMID: 23388448
Insulin-like growth factor 2; paternal obesity; DNA methylation; genomic imprinting
4.  Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop 
Nucleic Acids Research  2013;41(4):2171-2179.
Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an ∼1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions ∼5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3′ untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms.
doi:10.1093/nar/gks1355
PMCID: PMC3575839  PMID: 23295672
5.  Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach 
Molecular Therapy  2012;20(10):1953-1967.
Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.
doi:10.1038/mt.2012.117
PMCID: PMC3464631  PMID: 22760542
6.  The emerging role of epigenetic mechanisms in the etiology of neural tube defects 
Epigenetics  2011;6(7):875-883.
The molecular requirements for neural tube closure are complex. This is illustrated by the occurrence of neural tube defects (NTDs) in many genetic mouse mutants, which implicate a variety of genes, pathways and cellular functions. NTDs are also prevalent birth defects in humans, affecting around 1 per 1,000 pregnancies worldwide. In humans the causation is thought to involve the interplay of fetal genes and the effect of environmental factors. Recent studies on the etiology of human NTDs, as well as analysis of mouse models, have raised the question of the possible involvement of epigenetic factors in determining susceptibility. A consideration of potential causative factors in human NTDs must now include both alterations in the regulation of gene expression, through mutation of promoter or regulatory elements and the additional analysis of epigenetic regulation. Alterations in the epigenetic status can be directly modified by various environmental insults or maternal dietary factors.
doi:10.4161/epi.6.7.16400
PMCID: PMC3154428  PMID: 21613818
neural tube defects; diet; folic acid; epigenome; epigenetic regulation; methylation; chromatin; histones; acetylation
7.  The role of imprinted genes in humans 
Genome Biology  2011;12(3):106.
Detailed comprehensive molecular analysis using families and multiple matched tissues is essential to determine whether imprinted genes have a functional role in humans.
See research article: http://genomebiology.com/2011/12/3/R25
doi:10.1186/gb-2011-12-3-106
PMCID: PMC3129664  PMID: 21418582
8.  The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells 
Epigenetics  2011;6(1):52-62.
Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterize the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterized extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSC s exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.
doi:10.4161/epi.6.1.13361
PMCID: PMC3052914  PMID: 20864803
genomic imprinting; embryonic stem cells; mesenchymal stem cells; differentiation; methylation; epigenetic stability
10.  Human imprinted retrogenes exhibit non-canonical imprint chromatin signatures and reside in non-imprinted host genes 
Nucleic Acids Research  2011;39(11):4577-4586.
Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic ‘host’ gene. In the mouse, at least one transcript of the host gene is also subject to genomic imprinting. Human retrogene orthologues are imprinted and we reveal that human host genes are not imprinted. This coincides with genomic rearrangements that occurred during primate evolution, which increase the separation between the retrogene DMRs and the host genes. To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs. For the mouse retrogenes, the active mark H3K4me2 was associated with the unmethylated paternal allele, while the methylated maternal allele was enriched in repressive marks including H3K9me3 and H4K20me3. Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs. Finally, we show that the genes immediately flanking the host genes in mouse and human are biallelically expressed in a range of tissues, suggesting that these loci are distinct from large imprinted clusters.
doi:10.1093/nar/gkq1230
PMCID: PMC3113556  PMID: 21300645
12.  Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2 
The Journal of Clinical Investigation  2010;120(11):4102-4110.
Many common disorders of pregnancy are attributed to insufficient invasion of the uterine lining by trophoblast, fetal cells that are the major cell type of the placenta. Interactions between fetal trophoblast and maternal uterine NK (uNK) cells — specifically interactions between HLA-C molecules expressed by the fetal trophoblast cells and killer Ig-like receptors (KIRs) on the maternal uNK cells — influence placentation in human pregnancy. Consistent with this, pregnancies are at increased risk of preeclampsia in mothers homozygous for KIR haplotype A (KIR AA). In this study, we have demonstrated that trophoblast expresses both paternally and maternally inherited HLA-C surface proteins and that maternal KIR AA frequencies are increased in affected pregnancies only when the fetus has more group 2 HLA-C genes (C2) than the mother. These data raise the possibility that there is a deleterious allogeneic effect stemming from paternal C2. We found that this effect also occurred in other pregnancy disorders (fetal growth restriction and recurrent miscarriage), indicating a role early in gestation for these receptor/ligand pairs in the pathogenesis of reproductive failure. Notably, pregnancy disorders were less frequent in mothers that possessed the telomeric end of the KIR B haplotype, which contains activating KIR2DS1. In addition, uNK cells expressed KIR2DS1, which bound specifically to C2+ trophoblast cells. These findings highlight the complexity and central importance of specific combinations of activating KIR and HLA-C in maternal-fetal immune interactions that determine reproductive success.
doi:10.1172/JCI43998
PMCID: PMC2964995  PMID: 20972337
13.  Evaluation of Allelic Expression of Imprinted Genes in Adult Human Blood 
PLoS ONE  2010;5(10):e13556.
Background
Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults.
Methodology/Principal Findings
We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression.
Conclusions/Significance
We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression.
doi:10.1371/journal.pone.0013556
PMCID: PMC2958851  PMID: 21042416
14.  Telomeric NAP1L4 and OSBPL5 of the KCNQ1 Cluster, and the DECORIN Gene Are Not Imprinted in Human Trophoblast Stem Cells 
PLoS ONE  2010;5(7):e11595.
Background
Genomic imprinting of the largest known cluster, the Kcnq1/KCNQ1 domain on mChr7/hChr11, displays significant differences between mouse and man. Of the fourteen transcripts in this cluster, imprinting of six is ubiquitous in mice and humans, however, imprinted expression of the other eight transcripts is only found in the mouse placenta. The human orthologues of the latter eight transcripts are biallelically expressed, at least from the first trimester onwards. However, as early development is less divergent between species, placental specific imprinting may be present in very early gestation in both mice and humans.
Methodology/Principal Findings
Human embryonic stem (hES) cells can be differentiated to embryoid bodies and then to trophoblast stem (EB-TS) cells. Using EB-TS cells as a model of post-implantation invading cytotrophoblast, we analysed allelic expression of two telomeric transcripts whose imprinting is placental specific in the mouse, as well as the ncRNA KCNQ1OT1, whose imprinted expression is ubiquitous in early human and mouse development. KCNQ1OT1 expression was monoallelic in all samples but OSBPL5 and NAP1L4 expression was biallelic in EB-TS cells, as well as undifferentiated hES cells and first trimester human fetal placenta. DCN on hChr12, another gene imprinted in the mouse placenta only, was also biallelically expressed in EB-TS cells. The germline maternal methylation imprint at the KvDMR was maintained in both undifferentiated hES cells and EB-TS cells.
Conclusions/Significance
The question of placental specific imprinting in the human has not been answered fully. Using a model of human trophoblast very early in gestation we show a lack of imprinting of two telomeric genes in the KCNQ1 region and of DCN, whose imprinted expression is placental specific in mice, providing further evidence to suggest that humans do not exhibit placental specific imprinting. The maintenance of both differential methylation of the KvDMR and monoallelic expression of KCNQ1OT1 indicates that the region is appropriately regulated epigenetically in vitro. Human gestational load is less than in the mouse, resulting in reduced need for maternal resource competition, and therefore maybe also a lack of placental specific imprinting. If genomic imprinting exists to control fetal acquisition of maternal resources driven by the placenta, placenta-specific imprinting may be less important in the human than the mouse.
doi:10.1371/journal.pone.0011595
PMCID: PMC2904374  PMID: 20644730
15.  The Importance of Imprinting in the Human Placenta 
PLoS Genetics  2010;6(7):e1001015.
As a field of study, genomic imprinting has grown rapidly in the last 20 years, with a growing figure of around 100 imprinted genes known in the mouse and approximately 50 in the human. The imprinted expression of genes may be transient and highly tissue-specific, and there are potentially hundreds of other, as yet undiscovered, imprinted transcripts. The placenta is notable amongst mammalian organs for its high and prolific expression of imprinted genes. This review discusses the development of the human placenta and focuses on the function of imprinting in this organ. Imprinting is potentially a mechanism to balance parental resource allocation and it plays an important role in growth. The placenta, as the interface between mother and fetus, is central to prenatal growth control. The expression of genes subject to parental allelic expression bias has, over the years, been shown to be essential for the normal development and physiology of the placenta. In this review we also discuss the significance of genes that lack conservation of imprinting between mice and humans, genes whose imprinted expression is often placental-specific. Finally, we illustrate the importance of imprinting in the postnatal human in terms of several human imprinting disorders, with consideration of the brain as a key organ for imprinted gene expression after birth.
doi:10.1371/journal.pgen.1001015
PMCID: PMC2895656  PMID: 20617174
16.  Human Chromosome 7: DNA Sequence and Biology 
Scherer, Stephen W. | Cheung, Joseph | MacDonald, Jeffrey R. | Osborne, Lucy R. | Nakabayashi, Kazuhiko | Herbrick, Jo-Anne | Carson, Andrew R. | Parker-Katiraee, Layla | Skaug, Jennifer | Khaja, Razi | Zhang, Junjun | Hudek, Alexander K. | Li, Martin | Haddad, May | Duggan, Gavin E. | Fernandez, Bridget A. | Kanematsu, Emiko | Gentles, Simone | Christopoulos, Constantine C. | Choufani, Sanaa | Kwasnicka, Dorota | Zheng, Xiangqun H. | Lai, Zhongwu | Nusskern, Deborah | Zhang, Qing | Gu, Zhiping | Lu, Fu | Zeesman, Susan | Nowaczyk, Malgorzata J. | Teshima, Ikuko | Chitayat, David | Shuman, Cheryl | Weksberg, Rosanna | Zackai, Elaine H. | Grebe, Theresa A. | Cox, Sarah R. | Kirkpatrick, Susan J. | Rahman, Nazneen | Friedman, Jan M. | Heng, Henry H. Q. | Pelicci, Pier Giuseppe | Lo-Coco, Francesco | Belloni, Elena | Shaffer, Lisa G. | Pober, Barbara | Morton, Cynthia C. | Gusella, James F. | Bruns, Gail A. P. | Korf, Bruce R. | Quade, Bradley J. | Ligon, Azra H. | Ferguson, Heather | Higgins, Anne W. | Leach, Natalia T. | Herrick, Steven R. | Lemyre, Emmanuelle | Farra, Chantal G. | Kim, Hyung-Goo | Summers, Anne M. | Gripp, Karen W. | Roberts, Wendy | Szatmari, Peter | Winsor, Elizabeth J. T. | Grzeschik, Karl-Heinz | Teebi, Ahmed | Minassian, Berge A. | Kere, Juha | Armengol, Lluis | Pujana, Miguel Angel | Estivill, Xavier | Wilson, Michael D. | Koop, Ben F. | Tosi, Sabrina | Moore, Gudrun E. | Boright, Andrew P. | Zlotorynski, Eitan | Kerem, Batsheva | Kroisel, Peter M. | Petek, Erwin | Oscier, David G. | Mould, Sarah J. | Döhner, Hartmut | Döhner, Konstanze | Rommens, Johanna M. | Vincent, John B. | Venter, J. Craig | Li, Peter W. | Mural, Richard J. | Adams, Mark D. | Tsui, Lap-Chee
Science (New York, N.Y.)  2003;300(5620):767-772.
DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.
doi:10.1126/science.1083423
PMCID: PMC2882961  PMID: 12690205 CAMSID: cams403
17.  High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta 
BMC Genetics  2010;11:25.
Background
Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue.
Results
Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%).
Conclusions
Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.
doi:10.1186/1471-2156-11-25
PMCID: PMC2871261  PMID: 20403199
18.  Dynamic variation in allele-specific gene expression of Paraoxonase-1 in murine and human tissues 
Human Molecular Genetics  2008;17(21):3263-3270.
Differential allelic expression has been shown to be common in mice, humans and maize, and variability in the expression of polymorphic alleles has been associated with human disease. Here, we describe the differential expression pattern of Paraoxonase-1, a gene involved in lipid metabolism and implicated in the formation of atherosclerotic lesions. We measured the expression of the murine Paraoxonase-1 gene (Pon1) in livers at different stages of embryonic development using F1 hybrid crosses and quantified the transcriptional level of both parental alleles. Using human foetal tissues, we analysed the expression of the human orthologue (PON1) and found monoallelic or preferential allelic expression in 6/7 and 4/4 samples from liver and pancreas, respectively. We observed that Pon1 does not show a parent-of-origin preference in its allelic expression, but has dramatic variations in allele-specific expression occurring throughout development. This study has important repercussions in the analysis of haplotypes at disease loci, since it implies that the expression of polymorphic alleles can be unequal and dynamic.
doi:10.1093/hmg/ddn222
PMCID: PMC2566522  PMID: 18678600
19.  Tbx22null mice have a submucous cleft palate due to reduced palatal bone formation and also display ankyloglossia and choanal atresia phenotypes 
Human Molecular Genetics  2009;18(21):4171-4179.
Craniofacial defects involving the lip and/or palate are among the most common human birth defects. X-linked cleft palate and ankyloglossia results from loss-of-function mutations in the gene encoding the T-box transcription factor TBX22. Further studies show that TBX22 mutations are also found in around 5% of non-syndromic cleft palate patients. Although palate defects are obvious at birth, the underlying developmental pathogenesis remains unclear. Here, we report a Tbx22null mouse, which has a submucous cleft palate (SMCP) and ankyloglossia, similar to the human phenotype, with a small minority showing overt clefts. We also find persistent oro-nasal membranes or, in some mice a partial rupture, resulting in choanal atresia. Each of these defects can cause severe breathing and/or feeding difficulties in the newborn pups, which results in ∼50% post-natal lethality. Analysis of the craniofacial skeleton demonstrates a marked reduction in bone formation in the posterior hard palate, resulting in the classic notch associated with SMCP. Our results suggest that Tbx22 plays an important role in the osteogenic patterning of the posterior hard palate. Ossification is severely reduced after condensation of the palatal mesenchyme, resulting from a delay in the maturation of osteoblasts. Rather than having a major role in palatal shelf closure, we show that Tbx22 is an important determinant for intramembranous bone formation in the posterior hard palate, which underpins normal palate development and function. These findings could have important implications for the molecular diagnosis in patients with isolated SMCP and/or unexplained choanal atresia.
doi:10.1093/hmg/ddp368
PMCID: PMC2758147  PMID: 19648291
20.  Transcript- and tissue-specific imprinting of a tumour suppressor gene 
Human Molecular Genetics  2008;18(1):118-127.
The Bladder Cancer-Associated Protein gene (BLCAP; previously BC10) is a tumour suppressor that limits cell proliferation and stimulates apoptosis. BLCAP protein or message are downregulated or absent in a variety of human cancers. In mouse and human, the first intron of Blcap/BLCAP contains the distinct Neuronatin (Nnat/NNAT) gene. Nnat is an imprinted gene that is exclusively expressed from the paternally inherited allele. Previous studies found no evidence for imprinting of Blcap in mouse or human. Here we show that Blcap is imprinted in mouse and human brain, but not in other mouse tissues. Moreover, Blcap produces multiple distinct transcripts that exhibit reciprocal allele-specific expression in both mouse and human. We propose that the tissue-specific imprinting of Blcap is due to the particularly high transcriptional activity of Nnat in brain, as has been suggested previously for the similarly organized and imprinted murine Commd1/U2af1-rs1 locus. For Commd1/U2af1-rs1, we show that it too produces distinct transcript variants with reciprocal allele-specific expression. The imprinted expression of BLCAP and its interplay with NNAT at the transcriptional level may be relevant to human carcinogenesis.
doi:10.1093/hmg/ddn322
PMCID: PMC2666296  PMID: 18836209
21.  Somatically acquired hypomethylation of IGF2 in breast and colorectal cancer 
Human Molecular Genetics  2008;17(17):2633-2643.
The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2–5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.
doi:10.1093/hmg/ddn163
PMCID: PMC2515372  PMID: 18541649
22.  Genomic Imprinting of Dopa decarboxylase in Heart and Reciprocal Allelic Expression with Neighboring Grb10▿  
Molecular and Cellular Biology  2007;28(1):386-396.
By combining a tissue-specific microarray screen with mouse uniparental duplications, we have identified a novel imprinted gene, Dopa decarboxylase (Ddc), on chromosome 11. Ddc_exon1a is a 2-kb transcript variant that initiates from an alternative first exon in intron 1 of the canonical Ddc transcript and is paternally expressed in trabecular cardiomyocytes of the embryonic and neonatal heart. Ddc displays tight conserved linkage with the maternally expressed and methylated Grb10 gene, suggesting that these reciprocally imprinted genes may be coordinately regulated. In Dnmt3L mutant embryos that lack maternal germ line methylation imprints, we show that Ddc is overexpressed and Grb10 is silenced. Their imprinting is therefore dependent on maternal germ line methylation, but the mechanism at Ddc does not appear to involve differential methylation of the Ddc_exon1a promoter region and may instead be provided by the oocyte mark at Grb10. Our analysis of Ddc redefines the imprinted Grb10 domain on mouse proximal chromosome 11 and identifies Ddc_exon1a as the first example of a heart-specific imprinted gene.
doi:10.1128/MCB.00862-07
PMCID: PMC2223316  PMID: 17967881
23.  Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution  
PLoS Genetics  2007;3(5):e65.
Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.
Author Summary
Imprinted genes are expressed in a parent-of-origin manner, where one of the two inherited copies of the imprinted gene is silenced. Aberrations in the expression of these genes, which generally regulate growth, are associated with various developmental disorders, emphasizing the importance of their discovery and analysis. In this study, we identify a novel imprinted gene, named KLF14, on human Chromosome 7. It is predicted to bind DNA and regulate transcription and was shown to be expressed from the maternally inherited chromosome in all human and mouse tissues examined. Surprisingly, we did not identify molecular signatures generally associated with imprinted regions, such as DNA methylation. Additionally, the identification of numerous DNA sequence variants led to an in-depth analysis of the gene's evolution. It was determined that there is greater variability in KLF14 in the human lineage, when compared to other primates, with a significantly increased number of polymorphisms encoding for changes at the protein level, suggesting human-specific accelerated evolution. As the first example of an imprinted transcript undergoing accelerated evolution in the human lineage, we propose that the accumulation of polymorphisms in KLF14 may be aided by the silencing of the inactive allele, allowing for stronger selection.
doi:10.1371/journal.pgen.0030065
PMCID: PMC1865561  PMID: 17480121
24.  A Screen for Retrotransposed Imprinted Genes Reveals an Association between X Chromosome Homology and Maternal Germ-Line Methylation 
PLoS Genetics  2007;3(2):e20.
Imprinted genes undergo epigenetic modifications during gametogenesis, which lead to transcriptional silencing of either the maternally or the paternally derived allele in the subsequent generation. Previous work has suggested an association between imprinting and the products of retrotransposition, but the nature of this link is not well defined. In the mouse, three imprinted genes have been described that originated by retrotransposition and overlap CpG islands which undergo methylation during oogenesis. Nap1l5, U2af1-rs1, and Inpp5f_v2 are likely to encode proteins and share two additional genetic properties: they are located within introns of host transcripts and are derived from parental genes on the X chromosome. Using these sequence features alone, we identified Mcts2, a novel candidate imprinted retrogene on mouse Chromosome 2. Mcts2 has been validated as imprinted by demonstrating that it is paternally expressed and undergoes promoter methylation during oogenesis. The orthologous human retrogenes NAP1L5, INPP5F_V2, and MCTS2 are also shown to be paternally expressed, thus delineating novel imprinted loci on human Chromosomes 4, 10, and 20. The striking correlation between imprinting and X chromosome provenance suggests that retrotransposed elements with homology to the X chromosome can be selectively targeted for methylation during mammalian oogenesis.
Author Summary
The conventional view is that DNA carries all of our heritable information and our genes control development into adulthood. The discovery of epigenetics, a term coined to describe effects that are not coded for by DNA sequence, but can nonetheless affect our development and well-being, has added another layer of complexity to our understanding of genetics. One class of genes under epigenetic control are imprinted genes. Mammals inherit two copies of every gene, one from mother and one from father, and in most cases, both are active. However, for a small number of imprinted genes in mammals, only one is active, either the maternal or the paternal copy. Epigenetics amounts to a control system for switching genes on and off appropriately. We focus on a group of little-studied imprinted genes that share features that give clues to their evolutionary origins. These so-called “retrogenes” are protein-coding sequences of DNA that have undergone duplication and jumped into novel locations in the genome. Because of this, it is possible to determine where, and roughly when, many of the imprinted retrogenes originated. This provides an opportunity to study the molecular events that have generated imprinted genes during mammalian evolution.
doi:10.1371/journal.pgen.0030020
PMCID: PMC1796624  PMID: 17291163
25.  The growth hormone receptor gene deleted for exon three (GHRd3) polymorphism is associated with birth and placental weight 
Clinical Endocrinology  2012;76(2):236-240.
Context
Human growth hormone receptor (GHR) transcripts have two isoforms, full-length (GHRfl) or exon 3 deleted (GHRd3). An association of these isoforms has been found with small for gestational age (SGA) infants but does not influence adult height. The role of this polymorphism in the birth size spectrum in the general population is unclear.
Objective
To determine the association of maternal and infants GHR exon 3 polymorphism with antenatal growth, birth size and early postnatal growth in two large, normal white European birth cohorts.
Study design
Pregnant women from white European families were recruited by the University College London Foetal Growth Study (n = 774) and the Moore normal pregnancy cohort (n = 274). GHR variants, wild-type (fl) and deleted for exon 3 (d3) were analysed using multiplex PCR.
Results
There was a significant underrepresentation of infants wild-type fl/fl (36%) and overrepresentation of d3/d3 (14%) genotypes in the SGA infants within the cohorts (χ2 = 11·2, P = 0·003, df = 2). Fl/fl was overrepresented in large for gestational age (LGA) infants (χ2 = 6·1, P = 0·047, df = 2). There was a significant association of infants GHR isoforms with placental weight (P < 0·001) and birth weight standard deviation scores (P = 0·04) with the fl/fl genotype associated with a larger placental and birth weight. In multiple regression analysis, the GHR isoform type, maternal booking weight and parity influenced placental weight (R2 = 0·35; P < 0·001, df = 7). The GHR isoform type was not related to antenatal anthropometric measurements or growth in infancy.
Conclusion
These data suggest that the GHR isoforms are associated with placental and birth weight.
doi:10.1111/j.1365-2265.2011.04207.x
PMCID: PMC3321225  PMID: 21913951

Results 1-25 (28)