Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C/D box snoRNA cluster in Prader-Willi syndrome

Abstract

Prader-Willi syndrome (PWS) is a neurobehavioral disorder manifested by infantile hypotonia and feeding difficulties in infancy, followed by morbid obesity secondary to hyperphagia. It is caused by deficiency of paternally expressed transcript(s) within the human chromosome region 15q11.2. PWS patients harboring balanced chromosomal translocations with breakpoints within small nuclear ribonucleoprotein polypeptide N (SNRPN) have provided indirect evidence for a role for the imprinted C/D box containing small nucleolar RNA (snoRNA) genes encoded downstream of SNRPN. In addition, recently published data provide strong evidence in support of a role for the snoRNA SNORD116 cluster (HBII-85) in PWS etiology. In this study, we performed detailed phenotypic, cytogenetic, and molecular analyses including chromosome analysis, array comparative genomic hybridization (array CGH), expression studies, and single-nucleotide polymorphism (SNP) genotyping for parent-of-origin determination of the 15q11.2 microdeletion on an 11-year-old child expressing the major components of the PWS phenotype. This child had an ∼236.29 kb microdeletion at 15q11.2 within the larger Prader-Willi/Angelman syndrome critical region that included the SNORD116 cluster of snoRNAs. Analysis of SNP genotypes in proband and mother provided evidence in support of the deletion being on the paternal chromosome 15. This child also met most of the major PWS diagnostic criteria including infantile hypotonia, early-onset morbid obesity, and hypogonadism. Identification and characterization of this case provide unequivocal evidence for a critical role for the SNORD116 snoRNA molecules in PWS pathogenesis. Array CGH testing for genomic copy-number changes in cases with complex phenotypes is proving to be invaluable in detecting novel alterations and enabling better genotype-phenotype correlations.

Figure 1

Profile of patient at age 11 years. Note morbid obesity.

Figure 2

Characterization of microdeletion at 15q11.2 by microarray and FISH analyses. (a) Array CGH data (Roche NimbleGen 2.1 million CNV array) for proximal 15q11.2, and between SNRPN and UBE3A genes, showing loss in copy number for a segment between 22 774 847–23 007 783 (red bracket and vertical interrupted red lines). Tracks below the copy-number plot show distribution of segmental duplications followed by the track highlighting the gene content within interval. The SNORD116 and SNORD115 snoRNA clusters are highlighted by horizontal (red) brackets at the bottom of track. Results were visualized using Genoglyphix software (Signature Genomic Laboratories). The three reported deletions (including present case) shown at bottom of panel depicting relative genomic position and size. (b) Analysis by FISH with clone CTD-2283B2 (red signal) confirmed interstitial deletion at 15q11.2.

Figure 3

Breakpoint sequence and expression analysis. (a) Sequence analysis of ∼4 kb junction fragment revealed a 236 295 bp deletion. Insertion of a single base was also identified at the breakpoint. (b) Expression analysis by RT-PCR of whole blood (patient) or lymphoblast cell (controls) RNA for SNRPN, snoRNA clusters SNORD116, and UBE3A. GAPDH was used as internal RT-PCR control. (RT+: with reverse transcriptase; RT−: without reverse transcriptase). Patient (PWS) was negative for PCR product in RT+ lane for SNORD116 but positive for PCR product for all other tested exons.