Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) in Prader-Willi Syndrome

Soo-Jeong Kim¹, Jennifer L. Miller², Krista A. Schwenk^2,3, Christy L. Hollywood², Paul J. Kuipers²and Daniel J. Driscoll²

Departments of Psychiatry¹ and Pediatrics², and College of Education³, University of Florida

Introduction: Prader-Willi syndrome (PWS) is a rare genetic disorder caused by the functional absence of paternally expressed genes within the chromosome 15q11-q13 region via three main genetic mechanisms (i.e., deletion of paternally inherited genes, maternal uniparental disomy (UPD) and imprinting defect). The deletion group is further subdivided into two main subgroups: 1) type 1 deletions extend from breakpoint 1 (BP1) to breakpoint 3 (BP3) and 2) type 2 deletions that are approximately 500 kb smaller in size and extend from BP2 to BP3. Previous studies have found phenotypic differences between type 1 and type 2 deletions. Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) from MRC-Holland is a recently developed technique that can detect the copy number and DNA methylation status of genes (Nygren et al., NAR 2005; Proctor et al., Clin. Chem. 2006). We have applied this new methodology to our panel of patients with PWS. Our goal was: (1) to evaluate the efficacy of MS-MLPA as a diagnostic tool and (2) then to conduct genotype-phenotype correlations in our panel of patients.

Methods: The MS-MLPA kit contains 25 probes specific for sequences in the 15q11-q13 region which are designed to detect copy number and DNA methylation status of major genes in the region. Five out of these 25 probes also contain a DNA methylation sensitive restriction enzyme HhaI recognition site, thereby amplifying only methylated sequences after the digestion process. The amplified products were separated on the ABI 3730 automated sequencer and sized using the Peak Scanner Software v1.0 (www.appliedbiosystems.com).

Results: To date we have examined a total of 63 subjects with PWS: 51 patients with a deletion, 2 patients with a translocation and 10 patients with UPD, as well as 2 Angelman syndrome (AS) patients and 4 control subjects. MS-MLPA correctly identified the deleted and non-deleted PWS and AS subjects, as well as the normal controls. Among the 51 PWS patients with a deletion, 23 had a type 1 deletion and 23 had a type 2 deletion. Three subjects had a deletion between BP2 and BP4. Two subjects had a unique deletion: one subject was deleted from GCP5 to within the P(OCA2) gene and the other subject had a deletion of MKRN3 to within the ATP10A gene.

Conclusions: The MS-MLPA technique was able to provide significantly more information regarding the extent of the deletion in PWS subjects than the standard clinical Fluorescent In Situ Hybridization (FISH) technique. While microarray-based comparative genomic hybridization (CGH) generates more detailed information regarding the extent of the deletion, the high cost of this technique limits its feasibility in this context. Compared to array CGH, we found the MS-MLPA technique to be labor and cost effective. In addition, it also provides information regarding the DNA methylation status and correctly identified the PWS, AS and control subjects. The lack of probes proximal to BP1 and only one probe placed between BP3 and BP5 were the main limitations of MS-MLPA. In addition, we observed variability in the DNA methylation status for the NDN gene in some subjects. We are currently performing genotype-phenotype correlations in our patients with deletions.