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. 2019 Apr;7(4):e00575.
doi: 10.1002/mgg3.575. Epub 2019 Feb 21.

Analysis of the Prader-Willi syndrome imprinting center using droplet digital PCR and next-generation whole-exome sequencing

Samantha N Hartin  1 , Waheeda A Hossain  1 , David Francis  2 , David E Godler  2 , Sangjucta Barkataki  3 , Merlin G Butler  1
Affiliations

Analysis of the Prader-Willi syndrome imprinting center using droplet digital PCR and next-generation whole-exome sequencing

Samantha N Hartin et al. Mol Genet Genomic Med. 2019 Apr.

Abstract

Background: Detailed analysis of imprinting center (IC) defects in individuals with Prader-Willi syndrome (PWS) is not readily available beyond chromosomal microarray (MA) analysis, and such testing is important for a more accurate diagnosis and recurrence risks. This is the first feasibility study of newly developed droplet digital polymerase chain reaction (ddPCR) examining DNA copy number differences in the PWS IC region of those with IC defects.

Methods: The study cohort included 17 individuals without 15q11-q13 deletions or maternal disomy but with IC defects as determined by genotype analysis showing biparental inheritance. Seven sets of parents and two healthy, unrelated controls were also analyzed.

Results: Copy number differences were distinguished by comparing the number of positive droplets detected by IC probes to those from a chromosome 15 reference probe, GABRβ3. The ddPCR findings were compared to results from other methods including MA, and whole-exome sequencing (WES) with 100% concordance. The study also estimated the frequency of IC microdeletions and identified gene variants by WES that may impact phenotypes including CPT2 and NTRK1 genes.

Conclusion: Droplet digital polymerase chain reaction is a cost-effective method that can be used to confirm the presence of microdeletions in PWS with impact on genetic counseling and recurrence risks for families.

Keywords: Prader-Willi syndrome (PWS); droplet digital PCR; epimutation; imprinting center; microdeletion; whole-exome sequencing.

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Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
View of the chromosome 15 region involved in Prader–Willi syndrome (PWS) shown to scale with physical distance represented in million DNA base pairs (Mb) in size. (a) Chromosome 15 ideogram detailing banding pattern. Red box indicates the PWS region. Adapted from UCSC Genome Browser. (b) Genes in purple are expressed only from the paternal allele (MKRN3, MAGEL2, NECDIN, SNURF‐SNRPN, and SNORDs), genes in red only from the maternal allele (UBE3A and ATP10A) and genes in green show biallelic expression (GABRB3, GABRA5, and GABRG3, OCA2, HERC2, NIPA1, NIPA2, CYFIP1, and TUBGCP5). SNORD116 and SNORD115 are present in more repeat copies than indicated on the figure. BP1 to BP5 indicate positions of breakpoints on chromosome 15. (c) The imprinting center (IC) is shown below as a horizontal, blue line located between chr15:25.16‐25.20 Mb (hg19) as indicated by dotted red lines. The location of the three droplet digital polymerase chain reaction probes (IC2, IC1, and SNRPN exon 1) are shown
Figure 2
Figure 2
Proposed workflow for Prader–Willi syndrome (PWS)‐like phenotype referrals including Ch15 microdeletion droplet digital polymerase chain reaction (ddPCR) based analysis and next‐generation sequencing (NGS). Note: Diagnostic outcomes are highlighted in bold. *Consider other obesity‐related disorders; # PWS may present with atypical clinical features due to recessive disorder if mother is a carrier – consider WGS or WES. **If atypical PWS presentation is present, consider WGS or WES candidate gene studies
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