What are the indications for checking methylation status?

Indications for Checking Methylation Status

Methylation testing should be performed as the first-line diagnostic test for Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS), as it detects approximately 99% of cases, and is essential for confirming glioblastoma diagnosis in elderly or frail patients to guide temozolomide treatment decisions. 1, 2

Genetic Disorders Requiring Methylation Analysis

Prader-Willi and Angelman Syndromes

• Unexplained hypotonia with poor suck in infants requires immediate PWS methylation testing, as this is the most sensitive first-line diagnostic approach 2
• Feeding difficulties requiring assisted feeding methods should prompt methylation analysis for PWS 2
• Methylation analysis using Southern hybridization with SNRPN or PW71B probes confirms diagnosis when only maternal alleles are present (PWS) or only paternal alleles are present (AS) 1, 2
• If biparental inheritance is identified through methylation testing, both PWS and most identifiable causes of AS are ruled out 1

Fragile X Syndrome

• Methylation analysis reveals the degree of hypermethylation in full mutations and shows X-inactivation distribution in females with two distinguishable alleles 1
• Testing is indicated for individuals with intellectual disability, autism, learning disabilities, or family history of Fragile X syndrome 1
• Methylation status should NOT be used to predict severity in fetal or newborn cases, regardless of tissue source 1
• In chorionic villus samples obtained before 12.5 weeks gestation, methylation analysis is optional because the FMR1 region may not have complete methylation at this early stage 1, 3

Oncology Applications

Glioblastoma Management

• MGMT promoter methylation status must be determined in glioblastoma, particularly in elderly or frail patients, to guide temozolomide treatment decisions 1
• This is the single most important prognostic factor in the era of alkylating agent-based chemotherapy for gliomas 1
• Immunocytochemistry is NOT adequate for determining MGMT promoter methylation status; molecular methods are required 1

Brain Tumor Classification

• DNA methylation profiling serves as the gold standard for brain tumor classification, especially for medulloblastoma subtyping, which directly guides treatment decisions 4

Colorectal Cancer Screening

• SEPT9 promoter methylation testing is supported for colorectal cancer detection 4

Other Cancer Applications

• Cell-free DNA methylation analysis provides minimally invasive "liquid biopsy" for cancer detection and monitoring, with 64% sensitivity when combined with protein biomarkers 4
• Methylation-based assays are used for EGFR mutation detection in non-small cell lung cancer and PIK3CA mutation detection in breast cancer 4

Prenatal Diagnosis Scenarios

Specific Prenatal Indications

• When cytogenetic deletion is suspected on CVS or amniocentesis, perform FISH confirmation followed by methylation analysis if indicated 1
• When trisomy 15 mosaicism is detected on CVS or amniocentesis, perform PCR to detect uniparental disomy 15 1
• For imprinted genetic disorders detected by noninvasive prenatal screening (NIPS), methylation analysis is the appropriate diagnostic test, though methylation may not be complete at the time of CVS 1

Important Testing Considerations

Technical Requirements

• Methylation testing should be performed at academic tertiary centers with specialized expertise and proper quality control measures 4
• Standardized assays with high interlaboratory agreement are essential, particularly for MGMT promoter methylation 1
• Results can be affected by PCR bias, low read coverage, and SNPs at CpG sites, requiring specialized interpretation 4

Common Pitfalls to Avoid

• Do not use methylation patterns from blood samples to predict X-inactivation effects in other tissues for Fragile X syndrome 1
• Skewed X-inactivation can complicate detection of females with premutations using methylation-sensitive restriction enzymes 1
• Abnormal karyotypes (45,X or 47,XXY) can confound methylation analysis results and must be considered in interpretation 1
• In CVS samples, methylation associated with X-inactivation is usually absent, which is tissue-specific and should not be misinterpreted as abnormal 1, 3