Chromosome-wide identification of novel imprinted genes using microarrays and uniparental disomies

Abstract

Genomic imprinting refers to a specialized form of epigenetic gene regulation whereby the expression of a given allele is dictated by parental origin. Defining the extent and distribution of imprinting across genomes will be crucial for understanding the roles played by imprinting in normal mammalian growth and development. Using mice carrying uniparental disomies or duplications, microarray screening and stringent bioinformatics, we have developed the first large-scale tissue-specific screen for imprinted gene detection. We quantify the stringency of our methodology and relate it to previous non-tissue-specific large-scale studies. We report the identification in mouse of four brain-specific novel paternally expressed transcripts and an additional three genes that show maternal expression in the placenta. The regions of conserved linkage in the human genome are associated with the Prader-Willi Syndrome (PWS) and Beckwith-Wiedemann Syndrome (BWS) where imprinting is known to be a contributing factor. We conclude that large-scale systematic analyses of this genre are necessary for the full impact of genomic imprinting on mammalian gene expression and phenotype to be elucidated.

Figure 1

ROC curves for the SLR (solid), P-value (dashed), pSLR (finely dashed), uSLR (dotted) and vSLR (dash-dotted) differential expression measures. Each curve shows the TPR y-axis in relation to the FPR x-axis, estimated at various different decision thresholds for the respective measure and linearly interpolated in between the estimates. Labelled arrows point out thresholds of particular importance, specifically, the thresholds at which the estimated FPR was roughly 5% for all five measures, and/or, for uSLR and vSLR, thresholds that marked the end of steep increases in the TPR and therefore constituted particularly good trade-offs between TPR and FPR. The legend in the figure states for each measure the condition that a probe set needed to satisfy in order to be considered differentially expressed where r, s, t, u and v are the decision thresholds, and P and SLR are the P-value and SLR as computed by GCOS. d denotes Kronecker-δ, that equals 1 if the boolean expression in parenthesis (in our case unequality) is true, and that equals 0 otherwise. The uSLR consistently achieved the highest TPR for up to an FPR of 5%, closely followed by vSLR. Most significantly, P-value and SLR in isolation performed much worse as measures of differential expression than when combined in measures like uSLR.

Figure 2

Identification of novel paternally expressed transcripts on mouse proximal Chr 7. (A) The 2 Mb PWS/AS orthologous region with positions of the novel paternally expressed transcripts indicated^*. Genes are shown as open boxes with the relative transcriptional orientations defined by arrows. Blue, red or no colouring define paternal, maternal or biallelic expression respectively. (B) RT–PCR analysis of candidates identified on the proximal Chr 7 brain array in cDNA derived from patDp prox 7, matDp prox 7 and wild-type brain tissue. Controls for paternal (Snrpn), maternal (Grb10) and biallelic (Igf1r) expression are shown. The molecular weight marker is a 100 bp DNA ladder where the bright band corresponds to 500 bp. Samples treated with and without reverse transcriptase are indicated as + or −RT. ESTs AK080843, BB077283, BM117114 and AV328498 were paternally expressed. (C) Allele-specific RT–PCR analysis of proximal Chr 7 candidates. Newborn brain tissues with expressed SNPs were obtained from reciprocal crosses between M.m.musculus (B6) and M.m.castaneus (CAST) animals. cDNA fragments containing the SNPs were recovered by RT–PCR and direct sequencing to determine allele-specific expression.

Figure 3

Identification of novel maternally expressed transcripts on mouse distal Chr 7. (A) The ∼1 Mb BWS orthologous region shown with positions of the novel maternally expressed genes indicated^*. Regions of conserved linkage on human chromosomes are indicated by horizontal bars. Genes are shown as open boxes with the relative transcriptional orientations defined by arrows. Blue, red or no colouring define paternal, maternal or biallelic expression respectively. (B) Allele-specific RT–PCR analysis of distal Chr 7 candidates. Embryo and placenta (E13.5) tissues with expressed SNPs were obtained by performing reciprocal crosses between M.m.musculus (B6) and M.m.castaneus (CAST) animals. cDNA fragments containing the SNPs were recovered by RT–PCR then direct sequenced to determine allele-specific expression. Distal Chr 7 candidates; Dhcr7, Th and Ampd3 were maternally expressed in E13.5 placenta (lower panels) but biallelic in E13.5 embryo (middle panels). Ubiquitously imprinted (H19) and biallelic (Tnnt3) control genes are shown.

Figure 4

Imprinting analysis in human placenta. For DHCR7 two distinct polymorphisms, a G/A SNP at nucleotide 364 and a known T/C SNP at nucleotide 382 (rs1790334) in the DHCR7 cDNA were identified in two individuals by sequencing fetal DNA samples. For the AMPD3 analysis a T/A SNP at nucleotide 3160 in the AMPD3 cDNA was identified in one individual by sequencing fetal DNA samples. Allele-specific expression analysis in matched placenta cDNA showed the genes to be biallelic.