Abstract
Establishing or ruling out a molecular diagnosis of Prader–Willi or Angelman syndrome (PWS/AS) presents unique challenges due to the variety of different genetic alterations that can lead to these conditions. Point mutations, copy number changes, uniparental isodisomy (i-UPD) 15 of two subclasses (segmental or total isodisomy), uniparental heterodisomy (h-UPD), and defects in the chromosome 15 imprinting center can all cause PWS/AS. Here, we outline a combined approach using whole-exome sequencing (WES) and DNA methylation data with methylation-sensitive multiplex ligation-dependent probe amplification (MLPA) to establish both the disease diagnosis and the mechanism of disease with high sensitivity using current standard of care technology and improved efficiency compared to serial methods. The authors encourage the use of this approach in the clinical setting to confirm and establish the diagnosis and genetic defect which may account for the secondary genetic conditions that may be seen in those with isodisomy 15, impacting surveillance and counseling with more accurate recurrence risks. Other similarly affected individuals due to other gene disorders or cytogenetic anomalies such as Rett syndrome or microdeletions would also be identified with this streamlined approach.
Highlights
As reviewed previously, testing for Prader–Willi and Angelman syndromes (PWS/AS) has historically required a stepwise approach taking up valuable time and resources (e.g., Velinov et al, 2000; Martínez et al, 2006; Ramsden et al, 2010; Poole et al, 2013; Beygo et al, 2019; Butler et al, 2019a; Butler and Duis, 2020)
The AS Molecular Diagnostics heterozygosity (AOH) algorithm applied was able to identify which samples were due to segmental isodisomy 15 with the number and size of segments including their location and those with total isodisomy 15 due to errors in maternal meiosis I and meiosis II, respectively, this is important for genetic counseling and surveillance for other at-risk genetic conditions, if the mother or the father is a carrier of pathogenic autosomal-recessive gene variants on chromosome 15 in patients with these UPD subclasses (Driscoll et al, 1998; Bittel et al, 2006; Gold et al, 2018; Manzardo et al, 2018; Beygo et al, 2019; Butler et al, 2019a,b; Butler and Duis, 2020)
Females with Prader–Willi syndrome (PWS) may be at risk of having X-linked disorders due to skewed X chromosome inactivation from the trisomy 15 rescue event occurring during early embryonic development (e.g., Cassidy et al, 1992; Butler et al, 2007)
Summary
As reviewed previously, testing for Prader–Willi and Angelman syndromes (PWS/AS) has historically required a stepwise approach taking up valuable time and resources (e.g., Velinov et al, 2000; Martínez et al, 2006; Ramsden et al, 2010; Poole et al, 2013; Beygo et al, 2019; Butler et al, 2019a; Butler and Duis, 2020). Three different analytical modules are required for the WES analysis: sequence variant analysis including both the SNRPN and UBE3A genes, copy number analysis of the same and neighboring genes within the 15q11-q13 region, and absence of PWS and AS Molecular Diagnostics heterozygosity (AOH) analysis on chromosome 15q (see Figure 1). Sequence variant analysis from WES has been described on multiple occasions in the literature, while copy number analysis is more challenging. To address this challenge, the identification of the copy number variants (CNV) of three or more consecutive exons is utilized as an in-house developed method based on the comparison of normalized coverage to batch controls generating very high sensitivity. Microdeletions of the imprinting center can be detected in PWS (Tan et al, 2020)
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