Microarray Analysis of Gene/Transcript Expression in
Prader-Willi Syndrome: Deletion vs UPD

 Douglas Bittel, Nataliya Kibiryeva, Zohreh Talebizadeh, and Merlin G. Butler,  Section of Medical Genetics and Molecular Medicine, Children’s Mercy Hospitals and Clinics and University of Missouri - Kansas City School of Medicine, Kansas City, MO.

 Introduction: Prader-Willi syndrome (PWS), a contiguous gene disorder, is generally due to a paternally derived de novo 15q11-q13 deletion or maternal disomy 15 (UPD). The PWS critical region (PWSCR) is known to contain imprinted genes that are differentially expressed depending on the parent of origin. It is unclear how subtle changes in gene expression due to the loss of both imprinted genes and reduced expression of nonimprinted genes may lead to the clinical features seen in PWS. Therefore, in an effort to improve our understanding of gene expression within or close to the 15q11-q13 region, we constructed a custom cDNA microarray of 73 non-redundant sequences from this region.

Methods and Results: Our study consisted of age matched young adult males, six with PWS [3 with 15q11-q13 deletion (mean age 28 yrs.) and 3 with UPD (mean age 27 yrs.)] and three non-syndromic comparison males with obesity of unknown cause (mean age 26 yrs.). The custom array used for our analyses contained several genes known to be biallelically expressed (e.g., FIBRILLIN) and outside the PWSCR. The expression levels of these control genes isolated from actively growing lymphoblastoid cell lines were very similar in all subjects. Thirteen genes/transcripts showed no expression in the PWS deletion or UPD samples consistent with imprinting (paternally expressed). Fourteen of the genes/transcripts examined produced signal intensities inconsistent with equal expression from both alleles (biallelic) or exclusive expression from the paternal allele (maternally imprinted). These genes/transcripts with unusual expression patterns were divided into four groups. First, three transcripts had significantly less expression in the UPD cell lines than in either the deletion or control cell lines. These were all located outside the PWSCR. Since the expression from the UPD cell lines was reduced relative to both the control and deletion lines, the expression from the maternal allele must be significantly less than the expression from the paternal allele. The second group of transcripts consisted of five sequences including GABRA5 and GABRB3 genes from within the PWSCR, and the deletion lines had significantly less than half the control level of expression. Furthermore, signal intensities were significantly less in the UPD cell cultures compared to the control cell lines but greater than deletion lines. Taken together these data suggest paternal bias in the expression of these sequences. Third, two transcripts from just outside the PWSCR expressed at higher levels in deletion lines than either UPD or control lines. Fourth, four genes/transcripts, including UBE3A and ATP10C, appeared to have greater expression in the UPD lines than in either the control or deletion lines indicating maternal bias of expression.  UBE3A and ATP10 are known to be maternally expressed (paternally imprinted), although UBE3A only in the brain.

 Discussion: Our data suggest that the expression of genes and transcripts in and around the PWSCR is possibly influenced by chromatin structure and content, as well as, the imprinting center.  The dynamic interactions suggested by the microarray data reinforce the observations of the complex nature of expression in the 15q11-q13 region. Finally, we recognize that the targets applied to our microarray were isolated from lymphoblastoid cell cultures and gene expression in cell culture may not be in complete concordance with gene expression in brain tissue. Our results suggest candidate genes which may contribute to the differences observed between PWS subjects with deletions and UPD and warrants further investigation.

July 2003