The dilemma of diagnostic testing for Prader-Willi syndrome

Abstract

Although Prader-Willi syndrome (PWS) is a well-described clinical dysmorphic syndrome, DNA testing is required for a definitive diagnosis. A definitive diagnosis can be made in approximately 99% of cases using DNA testing; there are a number of DNA tests that can be used for this purpose, although there is no set standard algorithm of testing. The dilemma arises because of the complex genetic mechanisms at the basis of PWS, which need to be elucidated. To establish the molecular mechanism with a complete work up, involves at least 2 tests. Here we discuss the commonly used tests currently available and suggest a cost-effective approach to diagnostic testing.

Keywords: Cytogenetics; chromosome microarray; methylation.

Figure 1

The critical region for PWS on chromosome 15, with the main genes indicated. MKRN3 (or ZNF127) is a zinc finger protein, expressed only from the paternal chromosome; MAGEL2 is expressed only from the paternal chromosome mainly in the brain; NECDIN encodes a DNA binding protein; C15ORF2 is the open reading frame of the SNURF/SNRPN gene; MAGEL2, NDN and MKRN3 are all small intronless genes. Black lines and light blue ovals between BP2 and BP3 indicate imprinted genes in PWS, Black lines between BP1 and BP2 indicate non-imprinted genes and the 2 yellow circles are the maternally imprinted genes in Angelman syndrome. Exons 1-10 are within SNRPN; snoRNAs are here depicted pictorially. IPW, an RNA transcript lies within the snoRNA region, does not encode a protein but is paternally expressed only; SNORD116 also lies within the snoRNA region and is paternally expressed only. BP, breakpoint; Cen, centromere; tel, telomere.

Figure 2

The three molecular classes of PWS. Blue chromosome is paternal, pink is maternal. The yellow circle represents the 4–6 Mb critical region for PWS. The open circles indicate an active gene, the red cross an inactive (imprinted) gene. The black box indicates an IC defect. PWS, Prader-Willi syndrome; CI, imprinting centre.

Figure 3

A FISH picture of deletion: the deleted chromosome has no red signal under the centromere (green). The PML red signal is an extra control for the terminal end of chromosome 15. FISH, fluorescence in-situ hybridisation; FISH, fluorescence in-situ hybridisation; PML, promyelocytic leukemia.

Figure 4

Methylation-PCR DNA was modified by bisulfite treatment and the SNRPN exon 1 region was amplified by PCR. Normal control has both the maternal (313 bp) and paternal (221 bp) bands. Only the maternal band is visible in a Prader-Willi patient and the paternal band is present in an Angelman patient. PCR, polymerase chain reaction.

Figure 5

CGH chromosome microarray with type 1 deletion [between BP1–BP3 (GRCh37 chr15:22,749,354-28,438,266)], the CGH data showing Log2ratio “−1” between chromosome 15q11. 2q13. 1 regions indicating a heterozygous deletion. CGH + SNP chromosome microarray with type 2 deletion [between BP2-BP3 (GRCh37 chr15:23,619,912-28,438,266)], the CGH data showing Log2 ratio “−1” between chromosome 15q11. 2q13. 1 region indicating a heterozygous deletion. The SNP data also showed loss of one allele [displaying only the 0-uncut (AA) & 1-uncut allele (AB) track]. (A) CGH microarray-Type1 deletion; (B) CGH + SNP Microarray-Type2 deletion.

Figure 6

MS-MLPA showing normal control, PWS large common deletion, IC deletion and UPD.

Figure 7

Proposed testing strategy to diagnose and establish the molecular class in PWS using MS-MLPA as the first procedure.