When is a methylation test recommended?

When Methylation Testing is Recommended

Methylation testing should be ordered as first-line diagnostic testing for suspected Prader-Willi or Angelman Syndrome, for guiding temozolomide treatment in glioblastoma patients (particularly elderly or frail), for confirming Fragile X syndrome diagnosis, and when imprinted genetic disorders are detected on prenatal screening. 1, 2

Primary Diagnostic Indications

Genetic Disorders Requiring Methylation Analysis

Prader-Willi and Angelman Syndromes:

• Order methylation analysis as the first-line test when you observe unexplained hypotonia with poor suck, feeding difficulties requiring assisted methods, or clinical features suggesting these syndromes 2
• Methylation testing detects approximately 99% of Prader-Willi and Angelman Syndrome cases regardless of the underlying genetic mechanism (deletion, uniparental disomy, or imprinting defect) 1, 2
• Use Southern hybridization with methylation-sensitive SNRPN or PW71B probes to confirm diagnosis 2
• If only maternal methylation pattern is present, Prader-Willi Syndrome is confirmed; if only paternal pattern is present, Angelman Syndrome is confirmed 2

Fragile X Syndrome:

• Order methylation testing for individuals presenting with intellectual disability, autism, learning disabilities, or family history of Fragile X syndrome 1
• Methylation analysis distinguishes between methylation due to X-inactivation and hypermethylation of full mutations 3
• The term "methylation mosaic" or "incomplete methylation" applies when not all molecules in a full mutation are hypermethylated 3
• Males with unmethylated full mutations may have less severe phenotypes 3

Oncology Applications

Glioblastoma Management:

• Determine MGMT promoter methylation status in all glioblastoma patients, especially those aged ≥60 years or with poor performance status, to guide temozolomide treatment decisions 3, 1
• MGMT promoter methylation is the single most important prognostic factor in the era of alkylating agent-based chemotherapy for gliomas 1
• Patients with MGMT promoter-methylated tumors benefit substantially from temozolomide, while those without methylation show minimal benefit 3
• In elderly patients (≥60 years) with unmethylated MGMT promoter status, use hypofractionated radiotherapy alone; in those with methylated status, use temozolomide alone or combined with radiotherapy 3
• Use molecular methods rather than immunocytochemistry for determining MGMT promoter methylation status 1

Other Cancer Applications:

• DNA methylation-based assays support detection of EGFR mutations in non-small cell lung cancer, PIK3CA mutations in breast cancer, and SEPT9 promoter methylation for colorectal cancer screening 4
• DNA methylation profiling serves as the gold standard for brain tumor classification, particularly medulloblastoma subtyping 4

Prenatal Diagnosis Scenarios

Order methylation analysis when:

• Imprinted genetic disorders are detected by noninvasive prenatal screening (NIPS) 1
• Cytogenetic deletion involving chromosome 15 is suspected on CVS or amniocentesis 2
• Trisomy 15 mosaicism is detected prenatally 2
• A translocation involving chromosome 15 is identified prenatally 2

Emerging and Research Applications

Genetically Unsolved Cases

Consider genome-wide DNA methylation testing for:

• Individuals with neurodevelopmental presentations and congenital anomalies unexplained by conventional genetic testing 5
• Cases with variants of unknown clinical significance (VUS) in genes associated with syndromic conditions 5
• Genome-wide methylation analysis can concurrently detect 14 different syndromes with full accuracy and identify rare epi-variants that may explain clinical phenotypes 5

Important Testing Considerations

Quality and Interpretation:

• Methylation testing should be performed at academic tertiary centers with specialized expertise and proper quality control measures 1, 4
• Standardized assays with high interlaboratory agreement are essential for accurate MGMT promoter methylation testing 1
• Technical challenges include PCR bias, low read coverage, and SNPs at CpG sites, requiring specialized interpretation 4
• Harmonization in biospecimen collection, processing, and storage practices ensures accurate test results 4

Common Pitfalls to Avoid:

• Do not use methylation testing as a standalone test in oncology; it is most effective when combined with other biomarkers 4
• Do not interpret premutation carriers in Fragile X as simply "unaffected carriers"—they have risks for FXPOI (females) and FXTAS (both sexes) 3
• Do not assume methylation patterns in blood directly reflect brain methylation without validation, though emerging evidence suggests correlation for specific genes like BDNF 6