Abstract:
There is described a gene expression array comprising more than one imprinted gene and one or more control genes for the diagnosis of human epigenetic diseases resulting from assisted reproduction or the disruption.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a kit and method for detecting and/or screening for the presence of diseases, such as epigenetic disruption and associated diseases, and for assessing the risk of epigenetic disruption. 
       BACKGROUND 
       [0002]    Microarray analysis of gene expression typically yields expression data from thousands of genes, as the arrays that are used usually cover the whole or most of the genome (typically 12,000 to 30,000 genes on these arrays). This means that much data is generated that may not be of particular interest to the researcher or diagnostic team. In other words, the ‘extra’ data is not directly relevant to the project, hence time and effort is wasted performing complex data analysis. Therefore, by creating a focused array that is specific to a restricted number of genes, for example, genes that are grouped by sharing a common function, data handling is much easier and more straightforward. Furthermore, this would allow more rapid and efficient generation of results. 
         [0003]    Genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parent of origin specific manner. Forms of genomic imprinting have been demonstrated in insects, flowering plants and mammals, such as humans. 
         [0004]    In diploid organisms, such as humans, somatic cells possess two copies of the genome. Each autosomal gene is therefore represented by two copies, or alleles, with one copy inherited from each parent at fertilization. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. However, a small proportion (&lt;1%) of genes are imprinted, meaning that gene expression occurs from only one allele, i.e. the expressed allele is dependent upon its parental origin. 
         [0005]    Importantly, imprinted genes have diverse functions but exhibit a unique expression mechanism that makes them susceptible to being disrupted by cellular ‘environmental’ factors. This therefore makes them ideal as biomarkers or indicators of abnormality within a cell or tissue. Thus, imprinted genes can be markers of early cancer and induced cellular stresses observed in oocytes/embryos during in-vitro fertilization procedures. 
         [0006]    U.S. Pat. No. 6,235,474 describes a method and a kit for measuring abnormalities in imprinting in the development of cancer The imprinting can be abnormally on or can be abnormally off. In those cases where the particular gene that is being examined is normally imprinted, but in the disease state is abnormally not imprinted, the present invention is designed to detect the “loss of imprinting” thereby indicating that the disease may be present. However, the method/kit of U.S. Pat. No. &#39;474 measures the loss of imprinting for a single gene, therefore, whilst this may have some benefits, it clearly has severely limited practical use for the analysis of more complex gene systems. 
         [0007]    Epigenetic disruption in mammalian cell, such as a human cell, can lead either to an increase of expression of an imprinted gene or a decrease or even silencing of the imprinted gene. The processes involved may include relaxation of imprinting, gain of imprinting, promoter switch to a non-imprinted isoform, or from a non imprinted isoform to an imprinted one or another form of epigenetically-mediated silencing or activation of the imprinted gene. Furthermore, due to i) the reciprocal expression modes of certain imprinted genes (e.g. H19, IGF2), ii) the fact that expression of certain imprinted genes is accompanied or regulated by expression of imprinted antisense transcripts, and iii) that imprinted genes work in networks, it is likely that epigenetic disruption in a cell will be reflected by expression changes of more than one imprinted gene transcript. Tables I, II and III detail the aberrant regulation and expression of imprinted gene transcripts following assisted reproductive techniques (gametes and embryos), in embryonic stem cells and following somatic cell nuclear transfer to produce cloned embryos. It therefore follows that if epigenetic disruption is induced for example in embryos by a given Assisted Reproductive Technology (ART) procedure, then the associated up-regulation or down-regulation of imprinted gene expression will be detectable using a sufficiently sensitive imprinted gene expression array that is adequately controlled. The epigenetically susceptible imprinted genes therefore will act as biomarkers of epigenetic disruption. 
         [0008]    We have now found that by utilising a set of specific imprinted genes in a controlled context of gene expression microarray we are able to provide a workable array and a method of diagnostic screening for disease states. 
       SUMMARY OF THE INVENTION 
       [0009]    Thus, according to a first aspect of the invention we provide a gene expression array comprising more than one imprinted gene and one or more control genes. The number of imprinted genes may vary, but generally, they will make up from 60 to 80% of the total array, preferably from 65 to 75%, e.g. 72%. The more than one imprinted genes may be selected from any known/predicted human imprinted genes across the imprinted regions on human chromosomes 1, 6, 7, 11, 12 13, 14, 15, 18, 19 and 20 and X. The choice of the imprinted gene(s) will be dependent upon, inter alia, the disease type intended to be diagnosed. Thus, for example, the imprinted gene may be selected for the diagnosis of human epigenetic diseases resulting from assisted reproduction or the disruption of imprinted gene expression caused by assisted reproduction and/or in vitro culture of mammalian gametes, embryos, stem cells, somatic cell lines and therapeutic stem cells, birth defects, mental retardation, obesity, gross motor disturbances, diabetes, molar pregnancy, disorders of genomic imprinting, other epigenetic diseases. 
         [0010]    When the gene expression array according to the invention is used for the diagnosis of disease resulting from assisted reproduction and/or in vitro culture, the imprinted gene may be more than one of H19, KCNQ1OT1, SNRPN, PEG1/MEST and IGF2. 
         [0011]    When the gene expression array according to the invention is used for the diagnosis of disease resulting from the disruption of imprinted gene expression in assisted reproduction and/or in vitro culture of mammalian gametes, embryos and/or stem cells, the imprinted gene may be more than one of H19, IGF2, MEG1, H19, KCNQ1OT1, SNRPN and PEG1/MEST. 
         [0012]    The one or more control genes, will preferentially be a non-imprinted control gene which is positioned within or adjacent to the imprinted region. In addition, other control genes which may be mentioned are “housekeeping” control genes, i.e. ubiquitously expressed genes. The number of control genes may vary, but generally, they will make up from 10 to 20% of the total array, preferably from 12 to 15%, e.g. 14%. The control genes will predominantly be made up of imprint region controls, preferably, the imprint region control will comprise 50% or more of the total control gene population. 
         [0013]    The array may optionally include one or more epigenetic regulator genes. When one or more epigenetic regulator genes is present, the number of optional epigenetic regulators may vary, but generally, they will make up from 5 to 15% of the total array, preferably from 8 to 12%, e.g. 10%. The number of control genes may also vary depending upon the presence of one or more epigenetic regulator genes. 
         [0014]    According to a further aspect of the invention we provide a method of detecting the presence of a disease in a subject which comprises obtaining a biological sample from the subject and screening the sample for abnormal imprinting or abnormal expression of epigenetic regulators in one or more gene simultaneously. The method of the invention preferably comprises the use of an array as hereinbefore described. 
         [0015]    An Imprinted Gene Expression Array according to the invention is advantageous for, inter alia, the following reasons:
       Imprinted gene expression is disrupted in cancer (Table I), human assisted reproduction (Table II), mammalian in vitro embryo manipulation and stem cell technologies (Table III), and epigenetic diseases.   Imprinted genes are uniquely susceptible to epigenetic disruption   Use imprinted genes themselves as Bio-markers of epigenetic disruption   Array can show epigenetic disruption across 11 or more chromosomes   Networked expression mode enhances likelihood of detection of epigenetic disruption. Specific inactivation of six imprinted genes has been reported in in vitro cell cultures exposed to cellular stress and this also occurs in tumours (Pantoja et al., 2005). Other papers support these observations of imprinted genes working in networks (Varrault et al., 2006). If the expression of imprinted gene Zac1 is inactivated, so are other imprinted genes Igf2, H19, Cdkn1c, and Dlk1.   Array features transcripts from complex imprinted loci, (antisense transcripts etc.)   100 genes only on array-reduces complexity of bioinformatics   Bespoke array-allows expression analysis of all imprinted genes (n=70) arising from single embryo or small biopsy (less than 100 cells)   Alternatives such as bisulphite sequencing analysis limited to one gene/region for similar numbers of cells.   The pattern of disrupted imprinted gene expression revealed by the array will reveal the likely origin of the causative epigenetic mutation.       
 
         [0026]    An Imprinted Gene Expression Array according to the invention has utility both as a research tool and has commercial applications/markets:
       i) ART Industry. Screening for safety of existing and emerging human Assisted Reproductive Technologies to minimize epigenetic dysregulation as represented by a disruption of genomic imprinting following in vitro fertilization treatment and thus to minimize downstream epigenetic diseases. The array has been used successfully on single human embryos and oocytes—an essential requirement for this analysis. At this level of sensitivity, alternative techniques (e.g. bisulphite sequencing) can only assess epigenetic regulation at single or a restricted number of loci.   ii) Screening Human Stem Cells (embryonic, germ and adult types) and therapeutic differentiated cell derivatives thereof to ensure that disruption of imprinting (for example, deregulation of imprinting induced by extended cell-culture), is not a feature of the stem cell derivatives that are transplanted into the human body.   iii) Diagnostic or Research Product. For studying human imprinted diseases and epigenetic disorders, epigenetic mechanisms. For diagnostic investigation of disruption of imprinted gene expression in human imprinted diseases (Prader-Willi syndrome, Beckwith-Wiedemann syndrome, Angelman syndrome, Wilm&#39;s Tumour, Silver-Russell syndrome, Transient Neonatal Diabetes Mellitus, Rett&#39;s Syndrome, Molar pregnancies). Use in conjunction with traditional genomic diagnostic techniques to identify the genetic/epigenetic lesion and the genes that are affected in the patient.   iv) General Research Tool. Analysis of epigenetic regulation in experimental cells/animals, stem cell lines and, somatic cell lines and/or therapeutic stem cells that have undergone specific treatments/interventions that may disrupt epigenetic systems/imprinted gene expression (e.g. gene knockouts experiments, RNA interference experiments, somatic cell nuclear transfer (cloning) or similar procedures).       
 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0031]    The invention will now be described, by way of example only, with reference to the accompany tables and figures. 
       Example 1 
     Description of Array 
       [0032]      FIG. 1  is an imprinted gene array according to the invention. The array is a focused oligonucleotide microarray, specifically representing the human imprinted genes. The array features 60-mer oligonucleotides designed to be specific for these genes. 
       Example 2 
       [0033]    Somatic Tissue Testing. The array has been extensively tested in mRNAs derived from a range of human somatic tissue. Testis and ovary and brain as compared to a reference multi-tissue mRNA sample are shown in  FIG. 2 . Three differentially expressed genes are arrowed in the testis sample as examples. 
       Example 3 
     Data Output 
       [0034]    An expected straight line graph is produced when analyzing the background-subtracted signal ratio from two differently labelled tissues hybridised on the array  FIG. 3  (left). The differentially expressed genes are identified in  FIG. 3  (right). 
       Example 4 
     Testing in Single Human Oocytes and Preimplantation Embryos 
       [0035]    Over 40 single human oocytes or preimplantation embryos of various stages have individually been tested successfully on the array, proving the device is sufficiently sensitive to detect imprinted gene expression with a high degree of sensitivity. Dynamic regulation of imprinted gene expression are observed for these stages of development. Between 25 and 30 imprinted genes are expressed in the human blastocyst ( FIG. 4 ). 
       Example 5 
     Verification of Differentially Expressed Genes by Semi-Quantitative PCR 
       [0036]    The imprinted gene microarray was used in a series of replicate experiments (including dye-swaps) comparing expression in brain versus mixed tissue. Genes that were revealed to be consistently differentially expressed between brain and mixed tissue samples were subjected to verification by Semi-Quantitative RT-PCR. Results were consistent with array data. Four of the genes up-regulated in mixed tissue relative to brain are shown below. ASLC22A18, PAR1 and TRPM5 are imprinted genes ( FIG. 5 ). 
       Example 6 
     Tables 
       [0037]    Table I: illustrates human imprinted genes affected by Assisted Reproduction and resulting diseases as determined by the analysis of the use of ART in imprinted disease cohorts.
 
Table II: illustrates experimental data for mammalian imprinted genes and epigenetic regulators that are affected by assisted reproduction, and the underlying cause or molecular defects incurred.
 
Table III: illustrates data for disruption of imprinted gene expression in cloned assisted reproduction of mammalian, such as human, embryos, and stem cells that are derived somatic cell nuclear transfer and related technologies and the underlying cause or molecular defects incurred.
 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Human Imprinted Genes Affected by Assisted Reproduction, and 
               
               
                 Resulting Diseases. 
               
             
          
           
               
                   
                   
                   
                 Imprinted 
                   
               
               
                   
                   
                   
                 Gene(s) 
                   
               
               
                   
                 Imprinted Disease 
                 ART procedure 
                 affected 
                 Type of genetic defect 
               
               
                   
               
               
                 IMPRINTING  
                 Beckwith- 
                 IVF and ICSI 
                 KCNQ1OT1, 
                 KCNQ1OT1 
               
               
                 DISORDERS 
                 Wiedemann 
                   
                 H19 
                 Hypomethylation, H19 
               
               
                   
                 syndrome (BWS) 
                   
                   
                 hypermethylation 
               
               
                   
                 (BWS) 
                 IVF and ICSI 
                 KCNQ1OT1 
                 LIT1 Hypomethylation 
               
               
                   
                 BWS 
                 IVF and ICSI 
                 KCNQ1OT1 
                 LIT1 Hypomethylation 
               
               
                   
                 BWS 
                 ICSI 
                 n.d 
                 n.d 
               
               
                   
                 BWS 
                 IVF and ICSI 
                 n.d 
                 n.d 
               
               
                   
                 BWS 
                 IVF and ICSI 
                 n.d 
                 n.d 
               
               
                   
                 BWS 
                 IVF 
                 n.d 
                 n.d 
               
               
                   
                 BWS 
                 IVF 
                 n.d 
                 n.d 
               
               
                   
                 BWS 
                 ART 
                 KCNQ1OT1 
                 loss of methylation at 
               
               
                   
                   
                   
                 and multiple 
                 KCNQ1OT1 and other 
               
               
                   
                   
                   
                 imprinted loci 
                 defects 
               
               
                   
                   
                   
                 outside of 
                   
               
               
                   
                   
                   
                 chromosome 
                   
               
               
                   
                   
                   
                 11 
                   
               
               
                   
                 Not determined, but 
                 ICSI/ROSI) 
                 H19 
                 Aberrant methylation of 
               
               
                   
                 BWS may be 
                 using sperm 
                   
                 H19 in oligozoospermic 
               
               
                   
                 implicated 
                 from 
                   
                 males 
               
               
                   
                   
                 oligozoospermic 
                   
                   
               
               
                   
                   
                 males. 
                   
                   
               
               
                   
                 Angelman syndrome 
                 ICSI 
                 SNRPN 
                 (epimutation in 
               
               
                   
                 (AS) 
                   
                   
                 imprinting control region) 
               
               
                   
                 AS 
                 ICSI 
                 SNRPN 
                 (epimutation in 
               
               
                   
                   
                   
                   
                 imprinting control region) 
               
               
                   
                 Silver-Russell 
                 IVF 
                 PEG1/MEST 
                   
               
               
                   
                 syndrome 
               
               
                   
               
             
          
         
       
     
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                 TABLE II 
               
             
             
               
                   
               
               
                 Disruption of Imprinted Gene Expression and, epigenetic regulation 
               
               
                 following Assisted Reproduction of Mammalian Gametes and Embryos. 
               
             
          
           
               
                   
                   
                   
                 Affected Gene 
                   
               
               
                   
                   
                   
                 or Epigenetic 
                   
               
               
                 Cell Type 
                 ART Technique 
                 Species 
                 Mark 
                 Comments 
               
               
                   
               
               
                 PREIMPLANTATION  
                 Culture Media 
                 Mouse 
                 H19, Igf2 
                 Aberrant expression due to presence of FCS in 
               
               
                 EMBRYOS 
                   
                   
                   
                 M16 culture medium. 
               
               
                   
                 Culture Media 
                 Mouse 
                 H19 
                 Loss of H19 imprinting in extraembryonic 
               
               
                   
                   
                   
                   
                 tissues 
               
               
                   
                 Culture. Media 
                 Mouse 
                 H19 
                 Loss of H19 methylation upon culture in 
               
               
                   
                   
                   
                   
                 Whitten&#39;s medium. 
               
               
                   
                 Culture Media 
                 Mouse 
                 Igf2 
                 Aberrant expression bias to maternal allele in 
               
               
                   
                   
                   
                   
                 preimplantation embryo 
               
               
                   
                 Culture Media 
                 Mouse 
                 H19 
                 High levels of ammonium causes aberrant 
               
               
                   
                   
                   
                   
                 expression of H19 
               
               
                   
                 Culture Media 
                 Mouse 
                 Igf2, Meg1 and 
                 Reduced expression of three imprinted genes 
               
               
                   
                   
                   
                 Peg1 
                 after culture with FCS 
               
               
                   
                 Culture Media 
                 Mouse 
                 H19, Igf2 
                 Quinn&#39;s medium causes aberrant H19 
               
               
                   
                   
                   
                   
                 expression in embryos, aberrant H19 and Igf2 
               
               
                   
                   
                   
                   
                 in ES cells 
               
               
                   
                 In vitro culture 
                 Sheep 
                 Igf2r 
                 Reduced expression and methylation of Igf2r 
               
               
                   
                   
                   
                   
                 in a Large Offspring Syndrome model 
               
               
                   
                 In vitro 
                 Cow 
                 Igf2, Igf2r 
                 Reduced expression in in vitro produced 
               
               
                   
                 development 
                   
                   
                 embryos compared to in vivo embryos 
               
               
                   
                 In vitro culture 
                 Mouse 
                 Dnmt1 
                 Increased Dnmt1 expression in in vitro 
               
               
                   
                   
                   
                   
                 produced blastocysts 
               
               
                   
                 Culture Media 
                 Cow 
                 Dnmt1, Mash2 
                 Increased Dnmt1 expression, decreased Mash2 
               
               
                   
                   
                   
                   
                 in in vitro produced blastocysts 
               
               
                   
                 In vitro 
                 Mouse, 
                 DNA 
                 Increased DNA methylation compared to in 
               
               
                   
                 development 
                 rat 
                 methylation 
                 vivo embryos 
               
               
                   
                 Culture Media 
                 Cow 
                 Dnmt3a, Igf2r 
                 Upregulated expression of Dnmt3a and Igf2r 
               
               
                   
                   
                   
                   
                 in CR1aa and KSOMaa respectively 
               
               
                 SPERM 
                 ROSI 
                 Mouse 
                 H19 
                 Altered expression of H19 in extraembryonic 
               
               
                   
                   
                   
                   
                 tissues 
               
               
                   
                 ROSI 
                 Mouse 
                 Histone 
                 Significant difference in DNA methylation 
               
               
                   
                   
                   
                 methylation 
                 and histone methylation dynamics compared 
               
               
                   
                   
                   
                   
                 to ICSI embryos 
               
               
                   
                 ROSI 
                 Mouse 
                 DNA 
                 Abnormal localization of methylated 
               
               
                   
                   
                   
                 methylation 
                 chromatin in male pronucleus in fertilised 
               
               
                   
                   
                   
                   
                 oocytes derived from ROSI 
               
               
                 OOCYTES 
                 In vitro growth of 
                 Mouse 
                 Igf2r, Peg1, H19 
                 Loss of methylation at Igf2R and Peg1. Gain 
               
               
                   
                 follicles 
                   
                   
                 of methylation at H19 
               
               
                   
                 In vitro maturation 
                 Mouse 
                 Peg1 
                 In vitro culture for 8 h, Peg1/Mest DMR 
               
               
                   
                   
                   
                   
                 becomes fully methylated. Demethylation 
               
               
                   
                   
                   
                   
                 may occur after culture for 28 hrs in vitro 
               
               
                   
                 In vitro maturation 
                 Human 
                 HI 9 
                 Abnormal methylation at H19 locus 
               
               
                   
                 Superovulation 
                 Mouse 
                 DNA 
                 Global methylation abnormalities 
               
               
                   
                   
                   
                 methylation 
                   
               
               
                   
                 Superovulation 
                 Human 
                 H19, PEG1 
                 Aberrant gain of methylation at H19. loss of 
               
               
                   
                   
                   
                   
                 methylation at the PEG1 gene 
               
               
                   
                 Cause not 
                 Human 
                 KvDMR1 
                 Failure to establish methylation imprint at 
               
               
                   
                 identified 
                   
                   
                 KvDMR1 in a MI oocyte 
               
               
                   
               
             
          
         
       
     
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                 TABLE III 
               
             
             
               
                   
               
               
                 Disruption of Imprinted Gene Expression in Cloned Mammalian 
               
               
                 Embryos, and Embryonic Stem Cells. 
               
             
          
           
               
                   
                   
                 IMPRINTED 
                   
               
               
                   
                 Technique/ 
                 Gene(s) 
                   
               
               
                   
                 study focus 
                 affected 
                 Comments 
               
               
                   
               
               
                 Cloned 
                 SCNT 
                 Imprinting 
                 Disruptions in total transcript abundance 
               
               
                 Preimplantation 
                   
                 control regions 
                 and allele specificity of expression for five 
               
               
                 embryos 
                   
                 of the H19 and 
                 imprinted genes in cloned blastocysts. Loss 
               
               
                   
                   
                 Snprn genes 
                 of allele-specific DNA methylation at H19 
               
               
                   
                   
                   
                 and Snprn genes 
               
               
                   
                 SCNT 
                 U2af1-rs1, 
                 Loss of methylation and biallelic expression 
               
               
                   
                   
                 Igf2, H19, 
                 of U2af1-rs1, maternal methylation and 
               
               
                   
                   
                 Igf2r 
                 predominantly maternal expression of Igf2, 
               
               
                   
                   
                   
                 and biallelic methylation and expression of 
               
               
                   
                   
                   
                 Igf2r. Biallelic repression of H19 
               
               
                   
                 SCNT 
                 Genome-wide 
                 Aberrant methylation reprogramming in 
               
               
                   
                   
                 study 
                 cloned preimplantation embryos. 
               
               
                 Embryonic 
                 Cell culture  
                 IGF2, H19 
                 Aberrant methylation and aberrant 
               
               
                 Stem Cells 
                 (after SCNT) 
                   
                 (increased) expression of IGF2 and H19 
               
               
                   
                   
                   
                 associated with serum depletion and high 
               
               
                   
                   
                   
                 density culture 
               
               
                   
                 SCNT 
                 IGF2, H19 
                 Significant variation in expression of H19 
               
               
                   
                   
                   
                 and IGF2 in ES cells and also in the 
               
               
                   
                   
                   
                 placentas of foetuses derived from these ES 
               
               
                   
                   
                   
                 cells 
               
               
                   
                 SCNT 
                 H19, IGF2, 
                 Altered expression and methylation of H19 
               
               
                   
                   
                 IGF2R 
                 IGF2 AND IGF2R in cloned embryos 
               
               
                   
                   
                   
                 derived from ES cells