Whole genome microarray analysis of gene expression in Prader-Willi syndrome

Abstract

Prader-Willi syndrome (PWS) is caused by loss of function of paternally expressed genes in the 15q11-q13 region and a paucity of data exists on transcriptome variation. To further characterize genetic alterations in this classic obesity syndrome using whole genome microarrays to analyze gene expression, microarray and quantitative RT-PCR analysis were performed using RNA isolated from lymphoblastoid cells from PWS male subjects (four with 15q11-q13 deletion and three with UPD) and three age and cognition matched nonsyndromic comparison males. Of more than 47,000 probes examined in the microarray, 23,383 were detectable and 323 had significantly different expression in the PWS lymphoblastoid cells relative to comparison cells, 14 of which were related to neurodevelopment and function. As expected, there was no evidence of expression of paternally expressed genes from the 15q11-q13 region (e.g., SNRPN) in the PWS cells. Alterations in expression of serotonin receptor genes (e.g., HTR2B) and genes involved in eating behavior and obesity (ADIPOR2, MC2R, HCRT, OXTR) were noted. Other genes of interest with reduced expression in PWS subjects included STAR (a key regulator of steroid synthesis) and SAG (an arrestin family member which desensitizes G-protein-coupled receptors). Quantitative RT-PCR for SAG, OXTR, STAR, HCRT, and HTR2B using RNA isolated from their lymphoblastoid cells and available brain tissue (frontal cortex) from separate individuals with PWS and control subjects and normalized to GAPD gene expression levels validated our microarray gene expression data. Our analysis identified previously unappreciated changes in gene expression which may contribute to the clinical manifestations seen in PWS.

Fig. 1

Heat maps of microarray data of groups of genes from the three comparison males followed by the four PWS deletion subjects and three PWS UPD subjects. A: 900 probes chosen at random from the 23,383 probes meeting inclusion criteria, (B) neurodevelopment and function genes, (C) lipid metabolism and/or obesity genes, (D) serotonin receptor genes, and (E) GABA receptor subunit genes.

Fig. 2

Representative examples of quantitative RT-PCR assays using RNA from lymphoblastoid cells from three controls and four PWS males and the frontal cortex from a separate group of three controls and three PWS females from the NIH brain and tissue banks for both STAR and HTR2B clearly showing reduced gene expression in the PWS subjects in both lymphoblastoid cells and frontal cortex. The cycle threshold (C_T) for sample comparison was set at the narrowest portion of the logarithmic phase of PCR cycle amplification at which the signal intensity exceeds the detection threshold. Reported C_T values were normalized relative to GAPD expression (see Methods for description of normalization process) to account for quantity and quality differences between RNA samples. A: Representative quantitative RT-PCR with primers specific to STAR using RNA from lymphoblastoid cells. The GAPD normalized mean C_Ts were 22.7 and 26.6 for obese and PWS subjects respectively which represents a 14.9-fold reduction in the PWS subjects. B: Representative quantitative RT-PCR with primers specific to STAR using RNA from the frontal cortex. The GAPD normalized mean C_Ts were 23.3 and 27.4 for comparison and PWS subjects respectively which represents a 17.2-fold reduction in the PWS subjects. C: Representative quantitative RT-PCR with primers specific to HTR2B using RNA from lymphoblastoid cells. The GAPD normalized mean C_Ts were 18.5 and 21.4 for obese and PWS subjects respectively which represents a 7.5-fold reduction in the PWS subjects. D: Representative quantitative RT-PCR with primers specific to HTR2B using RNA from frontal cortex. The GAPD normalized mean C_Ts were 24.0 and 27.9 for comparison and PWS subjects respectively which represents a 14.9-fold reduction in the PWS subjects.