A newborn screening pilot study using methylation-sensitive high resolution melting on dried blood spots to detect Prader-Willi and Angelman syndromes

Abstract

Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct imprinted disorders characterized by genetic abnormalities at 15q11-q13. Early diagnosis of both syndromes provides improved treatment and accurate genetic counseling. Whole blood (WB) is the most common DNA source of many methodologies to detect PWS and AS, however, the need of WB makes a massive screening difficult in newborns due to economic and technical limitations. The aim of this study was to adapt a Methylation-sensitive High-Resolution Melting (MS-HRM) approach from dried blood spot (DBS) samples, assessing the different DNA isolation techniques and diagnostic performance. Over a 1-year period, we collected 125 DBS cards, of which 45 had already been diagnosed by MS-HRM (20 PWS, 1 AS, and 24 healthy individuals). We tested three different DBS-DNA extraction techniques assessing the DNA concentration and quality, followed by MS-HRM and statistical comparison. Each DBS-DNA extraction method was capable of accuracy in detecting all PWS and AS individuals. However, the efficiency to detect healthy individuals varied according to methodology. In our experience, DNA extracted from DBS analyzed by the MS-HRM methodology provides an accurate approach for genetic screening of imprinting related disorders in newborns, offering several benefits compared to traditional whole blood methods.

Figure. 1

Comparison of DNA quality between nucleic acid extraction methods. The DNA concentration (a), and DNA purity were assessed using 260/280 and 260/230 parameters (b,c). NS P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2

Variations of RPP38 amplification per DNA extraction method. (a) Comparative Ct amplification analysis ****P < 0.0001, **P < 0.01. Representative RPP38 amplification plots: (b) Qiagen-WB; (c) Qiagen-DBS; (d) Mem-DBS; (e) Chellex-DBS.

Figure 3

Dissociation curve analysis according to each DNA extraction method. (a–d) Normal methylation pattern, unmethylated paternal (78 °C), and methylated maternal alleles (82 °C) present. (e–h) Absence of paternal allele, only the maternal allele is present, confirming the PWS pattern. (i–l) Absence of the maternal allele, only the paternal allele is present, confirming AS pattern. Dissociation curve analysis per DNA extraction method.

Figure 4

Methodology of DNA methylation analysis for DBS. Step 1: Collect blood and fill the spots on filter papers; Step 2: Extract the DNA from each Guthrie cards and from Whole Blood; Step 3: Bisulfite treatment, converting unmethylated cytosine to uracil; Step 4: PCR amplification; Step 5: Allele discrimination through HRM methodology: Step 6: Results analysis and diagnostic.*It does not distinguish between the different molecular causes related to PWS and AS.