Methylation-Specific Testing

Methylation specific testing detects epigenetic changes: methylation of cytosine at CpG dinucleotides, which silences gene expression without altering the DNA sequence.

Methods are bisulphite conversion (unmethylated cytosines convert to uracil while methylated cytosines remain) followed by methylation specific PCR, MS-MLPA (methylation specific multiplex ligation dependent probe amplification, which detects copy number and methylation together), pyrosequencing, bisulphite sequencing, methylation arrays (450K and EPIC arrays covering 450 000 to 850 000 CpG sites), and digital PCR; enzymatic and long read methods avoid bisulphite damage.

Clinical uses are imprinting disorders, fragile X syndrome, cancer biomarkers, tumour classification and cell free DNA tests.

Imprinted gene disorders arise from loss of parent specific expression at defined loci: Prader-Willi syndrome (1 in 15 000; loss of paternal 15q11-q13 by deletion in 70%, maternal uniparental disomy in 25% and imprinting defect in 1 to 3%), Angelman syndrome (1 in 15 000; loss of maternal 15q11-q13: deletion 70%, paternal uniparental disomy 5%, imprinting defect 3%, UBE3A mutation 10%),

Beckwith-Wiedemann syndrome (1 in 10 000; 11p15 abnormalities), Silver-Russell syndrome (11p15 hypomethylation in 30 to 60%, maternal uniparental disomy 7 in 5 to 10%), transient neonatal diabetes (6q24) and Temple and Kagami-Ogata syndromes (chromosome 14).

In oncology, MGMT promoter methylation predicts response to temozolomide in glioblastoma, MLH1 promoter methylation separates sporadic from Lynch syndrome colorectal and endometrial cancers, SEPT9 methylation in plasma screens for colorectal cancer, and multi-cancer early detection tests use cell free DNA methylation patterns.

Indications. Neonatal hypotonia with poor feeding (Prader-Willi), developmental delay with seizures, ataxia, absent speech and happy demeanour (Angelman). Overgrowth, macroglossia, abdominal wall defect and neonatal hypoglycaemia (Beckwith-Wiedemann). Prenatal growth restriction with relative macrocephaly, feeding difficulty and body asymmetry (Silver-Russell).

Transient neonatal diabetes. Mismatch repair deficiency with loss of MLH1 on immunohistochemistry (to exclude Lynch syndrome). Glioblastoma (MGMT status for treatment). Fragile X syndrome (FMR1 promoter methylation in unclear repeat sizes). Selected cancer classification (central nervous system tumours, sarcomas) and cancer screening in research or approved settings.

Not for general screening for epigenetic age or lifestyle.

Specimen. Peripheral blood 2 to 5 mL in EDTA (DNA, with at least 1 microgram), saliva, or buccal swab for imprinting disorders. Tumour tissue (formalin fixed paraffin embedded with tumour content over 20 to 30%, marked by a pathologist), fresh frozen tissue, or plasma for cell free DNA (10 mL in cell stabilising tubes).

Parental samples for uniparental disomy analysis (microsatellite or SNP typing of trios). Bisulphite treatment requires good quality DNA, and degraded DNA reduces conversion efficiency.

Reference values. Normal pattern for the locus: for example, the 15q11-q13 SNRPN region has both methylated (maternal) and unmethylated (paternal) alleles in a 50:50 pattern. Cancer markers are reported as percentage methylation or positive or negative against a validated cut off (for MGMT, usually 10% or more methylated is positive, assay specific).

Interpretation.

• Prader-Willi syndrome: only the maternal (methylated) pattern at SNRPN, with absent paternal (unmethylated) band; confirmed in over 99% of cases. Further testing (microarray, FISH, microsatellite analysis) separates deletion, maternal uniparental disomy and imprinting defect, which have different recurrence risks (under 1% for deletion and uniparental disomy, up to 50% for imprinting centre microdeletion).
• Angelman syndrome: only the paternal (unmethylated) pattern; abnormal in about 80% of cases. A normal methylation result does not exclude Angelman syndrome (UBE3A mutation, 10 to 15%), so UBE3A sequencing follows.
• Beckwith-Wiedemann syndrome: loss of methylation at imprinting control region 2 (KCNQ1OT1) in 50%, gain of methylation at imprinting control region 1 (H19/IGF2) in 5%, paternal uniparental disomy of 11p15 in 20% (mosaic, so tissue dependent), CDKN1C mutations in 5%. Normal blood methylation does not exclude it because of mosaicism.
• Silver-Russell syndrome: hypomethylation at H19/IGF2 imprinting control region 1 in 30 to 60%, and maternal uniparental disomy 7 in 5 to 10%.
• Fragile X: methylation of the FMR1 promoter with an expanded CGG repeat over 200 (full mutation) causes silencing; MS-PCR resolves mosaics and premutation size ambiguity.
• MGMT promoter methylation in glioblastoma: methylated tumours respond better to temozolomide (median survival gain of 4 to 6 months), and the result directs treatment in older patients.
• MLH1 promoter hypermethylation with BRAF V600E: sporadic mismatch repair deficiency, and Lynch syndrome is unlikely; unmethylated MLH1 loss needs germline testing.
• Plasma SEPT9 and multi-cancer methylation tests: positive results require colonoscopy or imaging, and negative results do not exclude cancer.
• DNA methylation episignatures and classifiers define neurodevelopmental syndromes (CHARGE, Kabuki, Sotos) and tumour classes in specialist centres.

Critical values and findings needing immediate action.

• Neonate with hypotonia and feeding failure and abnormal SNRPN methylation: early nutritional support, growth hormone consideration, and prevention of obesity in infancy and childhood.
• Angelman syndrome diagnosis: seizure control, therapies and family counselling.
• Beckwith-Wiedemann syndrome diagnosis: tumour surveillance by abdominal ultrasound every 3 months until age 8 (alpha fetoprotein until age 4), and treatment of hypoglycaemia in newborns.
• Glioblastoma with unmethylated MGMT: discussion of alternative or investigational options with the oncology team.
• Lynch syndrome suspected on mismatch repair loss without MLH1 methylation: germline testing, colonoscopic surveillance and cascade testing.
• Prenatal diagnosis of an imprinting disorder: genetic counselling.

Interferences and pitfalls.

• Incomplete bisulphite conversion produces false methylation signals, and conversion controls are required; bisulphite degrades DNA, reducing sensitivity for low input samples.
• Methylation patterns are tissue specific, and mosaicism (especially in Beckwith-Wiedemann syndrome and Silver-Russell syndrome) can give normal blood results; testing of other tissues (buccal, skin) may be needed.
• Methylation testing does not detect the underlying mechanism; further tests are needed to define deletions, uniparental disomy or imprinting centre mutations for recurrence risk.
• Tumour content, necrosis, formalin fixation and heterogeneity affect MGMT and MLH1 results; different assays and cut offs are not interchangeable.
• Age, smoking, medications, cell type composition and environment alter methylation, so interpretation of cancer markers and epigenetic age needs validated references.
• Some commercial "epigenetic age" and lifestyle tests lack clinical validity.
• Blood cell free DNA methylation tests have lower sensitivity in early stage cancers (for example, 20 to 40% in stage I) and false positives can cause anxiety and further testing.
• Cost, technical expertise and access to specialist laboratories limit use.
• Counselling about recurrence risk requires a precise molecular classification, and families benefit from parental studies.
• Newer long read sequencing detects methylation directly and may replace bisulphite assays.

Special groups.

• Newborns and infants with hypotonia, hypoglycaemia or overgrowth: urgent testing because early management alters outcome.
• Children with developmental delay and seizures: Angelman syndrome and Rett syndrome (MECP2 sequencing) are in the differential.
• Prenatal diagnosis: uniparental disomy suspected from a trisomy rescue (chromosome 15, 7, 11, 14) or from a parental translocation.
• Assisted reproduction: slight increase in imprinting disorders (Beckwith-Wiedemann syndrome) reported, and counselling is needed.
• Oncology patients: MGMT, MLH1 and other epigenetic biomarkers in central nervous system, colorectal and endometrial cancers.
• Regions: availability is limited to genetics laboratories in tertiary centres, and samples are often referred.

Follow up tests. Chromosomal microarray, FISH, microsatellite or SNP analysis of parent child trios for uniparental disomy, MS-MLPA, UBE3A sequencing, MECP2 and other gene sequencing, imprinting centre deletion analysis, FMR1 repeat sizing, abdominal ultrasound and alpha fetoprotein surveillance in Beckwith-Wiedemann syndrome, endocrine assessment (growth hormone, thyroid, glucose) in Prader-Willi syndrome,

BRAF V600E and germline mismatch repair gene testing in suspected Lynch syndrome, and genetic counselling.