Table 2. Overview on the currently applied methods for 11p15.5 testing and their limitations.
| Suitability to detect | ||||||||
|---|---|---|---|---|---|---|---|---|
| Method | Loci per test | UPD | Epimutation | CNV | SNP | Suitable for prenatal testing | Average amount of DNA needed | Limitations |
| MS methods based on methylation-sensitive enzymes | ||||||||
| MS Southern blot | 1 | Y | Y | Y | N | (Y) | 5 μg | Large amounts of DNA, time-consuming. Main limitation is restriction site interrogation of epimutation at single CpGs within the DMRs Sensitivity is relatively low in comparison with the other techniques. No commercially available kit Only indirect discrimination of CNVs. In case of a positive result, further testing is required to discriminate the underlying change |
| MS-MLPA | Up to ~46a | Y | Y | Y | N | Y | 50–100 ng | Commercial kits. May have reduced sensitivity for detection of mosaic epimutation and UPD. SNVs can interfere. Dependent on the probe composition of the kit, a discrimination between the types of (epi)mutations and UPD can be possible |
| MS methods based on bisulfite treatment: these methods are not MS in sensu stricto, as they are based on primers equally amplifying unmethylated or methylated alleles | ||||||||
| MS PCR | 1 | Y | Y | Y | N | Y | 1 μg | No commercially available kit. Only indirect discrimination of CNVs. In case of a positive result, further testing is required to discriminate the underlying change |
| ASMM RTQ-PCR | 1 | Y | Y | N | N | Y | 150 ng | No commercially available kit. Only indirect discrimination of CNVs. In case of a positive result, further testing is required to discriminate the underlying change |
| MS-HRMA | 1 | Y | Y | Y | Y | Y | 1 μg | Does not examine discrete CpG sites but looks at regional profile. No commercially available kit Only indirect discrimination of CNVs. In case of a positive result, further testing is required to discriminate the underlying change |
| MS pyrosequencing | 1 | N | Y | Y | (Y) | Y | >100 ng | May have reduced sensitivity for detection of mosaic epimutation. Read-length limits analyzed CpGs per read. Only indirect discrimination of CNVs. In case of a positive result, further testing is required to discriminate the underlying change |
| MS-SNuPE | Up to 10 | Y | Y | Y | N | Y | 100 ng | No commercially available kit. Only indirect discrimination of CNVs. In case of a positive result, further testing is required to discriminate the underlying change |
| Non-MS tests for CNV and/or UPD detection | ||||||||
| Microsatellite analysis (STR) | 1 | Y | N | Y | N | Y | <20 ng per locus | DNA of at least one parent required; restricted to the detection of CNVs and UPD |
| qPCR | 1 | N | N | Y | N | Y | 200 ng | Only unbalanced alterations are detected. Flexible design targeting the region of interest; small CNVs can be detected |
| Molecular karyotyping (arrayCGH, SNP array) | Whole genome: depends on resolution | Y (SNP) N (CGH) | N | Y | N | Y | 250 ng | Only unbalanced alterations are detected Does not detect epimutations. No UPD detection by arrayCGH. Uneven probe coverage can leave CNVs undetected |
| DNA sequencing | Locus-specific | N | N | N | Y | Y | Depending on the size of the gene: ~60–200 ng | Only sequencing variants can be detected, other genomic variants including CNVs escape detection. In case of homozygosity of SNP large deletions should be considered |
Abbreviations: ASMM RTQ-PCR, allele-specific methylated multiplex real-time quantitative PCR; CHG, comparative genomic hybridization; CNV, copy number variation (deletions/duplications); MLPA, multiplex ligation probe-dependent amplification; MS, methylation-specific; MS-HRMA, MS-high-resolution melting analysis; MS-SNuPE, methylation-specific single-nucleotide primer extension; N, no; qPCR, quantitative PCR; SNP, single nucleotide variation – monogenic disease-causing variant; UPD, uniparental disomy; Y, yes.
a
The number of 46 probes refers to the conventional MLPA kits.