Trinucleotide Repeat Testing

Trinucleotide repeat disorders are caused by expansion of a short DNA sequence of three bases that is repeated many times in or near a gene. When the number of repeats goes beyond a threshold, the gene or its product is disrupted.

The disorders include Fragile X syndrome (CGG repeats in FMR1), Huntington disease (CAG in HTT), myotonic dystrophy type 1 (CTG in DMPK), several spinocerebellar ataxias (CAG), Friedreich ataxia (GAA in FXN), spinal and bulbar muscular atrophy (CAG in the androgen receptor), and dentatorubral pallidoluysian atrophy.

Repeats become longer when passed from parent to child, and longer repeats cause earlier and more severe disease, a pattern called anticipation. The parent who transmits the expansion matters: paternal transmission leads to larger expansions in Huntington disease, and maternal transmission leads to the large expansions in Fragile X syndrome and congenital myotonic dystrophy.

Testing uses PCR to amplify the repeat region, followed by capillary electrophoresis to size the repeats. Large expansions that cannot be amplified are detected by triplet primed PCR or Southern blot analysis, and methylation tests are used in Fragile X syndrome. Results take 1 to 3 weeks. A related type, hexanucleotide expansion of C9orf72 (GGGGCC), causes frontotemporal dementia and motor neurone disease, and it is tested in the same way.

These conditions are individually uncommon but collectively important. Fragile X is the most common inherited cause of intellectual disability (about 1 in 4000 males and 1 in 8000 females). Huntington disease affects about 3 to 10 in 100,000 people in populations of European origin, and it is less common in other regions.

Indications.

• Fragile X syndrome: unexplained intellectual disability, developmental delay, autism, a family history, premature ovarian insufficiency (premutation), and tremor and ataxia in an older carrier (FXTAS).
• Huntington disease: chorea, cognitive and psychiatric change with a family history, and predictive testing of at risk adults.
• Myotonic dystrophy type 1: myotonia, distal weakness, cataract, cardiac conduction defects, and congenital hypotonia in a newborn of an affected mother.
• Spinocerebellar ataxias and Friedreich ataxia: progressive ataxia of unknown cause, with or without a family history.
• C9orf72 repeat testing in frontotemporal dementia and motor neurone disease.
• Cascade testing of relatives, and prenatal diagnosis in at risk pregnancies.

Not indicated for predictive testing in children for adult onset diseases, unless there is a medical benefit in childhood.

Specimen requirements.

• EDTA blood (3 to 5 mL), or extracted DNA. Prenatal samples (chorionic villi or amniotic fluid) are tested for known familial expansions.
• Written consent, with a pre-test counselling visit, and for predictive testing a longer process with psychological support, a support person, and a wait before disclosure.
• Clinical details, family tree, and the age at onset in relatives.
• Family history of the repeat size, if known.

Normal appearances.

Normal alleles have repeat numbers under the disease thresholds, and they are stable when passed on. A normal result excludes the tested disorder in most cases, though not other causes of the same picture.

Interpretation.

• Fragile X (CGG repeats): normal 5 to 44, intermediate (grey zone) 45 to 54, premutation 55 to 200, and full mutation over 200 (with methylation of the promoter and silencing of FMR1). Premutation carriers are at risk of FXTAS and premature ovarian insufficiency, and women with premutations can have children with full mutations.
• Huntington disease (CAG repeats): normal 26 or fewer, intermediate 27 to 35 (not causing disease in the carrier, but can expand in the next generation), reduced penetrance 36 to 39, and full penetrance 40 or more. Longer repeats are linked to earlier onset.
• Myotonic dystrophy type 1 (CTG repeats): normal 5 to 34, premutation 35 to 49, mild disease about 50 to 150, classical disease 150 to 1000, and congenital disease over 1000.
• Spinocerebellar ataxias: the threshold depends on the type (for example, SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17), and expanded alleles correlate with age at onset.
• Friedreich ataxia (GAA repeats): about 66 or more on both alleles (usually 600 to 1200), and a minority have a point mutation on one allele.
• Spinal and bulbar muscular atrophy: 38 or more CAG repeats in men.
• C9orf72: over about 30 hexanucleotide repeats is pathogenic, with usually hundreds to thousands.
• Reduced penetrance and intermediate results need careful counselling, since they are not simple yes or no results.

Critical values and findings needing immediate action.

• A full mutation or expansion in a symptomatic child or adult: referral to a specialist team for management, genetic counselling and support.
• A positive predictive test in an unaffected person: immediate access to psychological support, since depression and suicide risk are raised in the period after disclosure.
• An expansion in a pregnant woman or in a foetus: urgent counselling about options.
• Premutation carriers with tremor, ataxia or ovarian insufficiency: referral to the right specialist.
• Women with myotonic dystrophy who are pregnant: obstetric and neonatal planning, since the baby may have congenital disease.

Limitations and artefacts.

• Sizing is imprecise for long repeats, and the number reported can differ slightly between laboratories and methods.
• Very large expansions fail to amplify, and a single band (apparent homozygosity) can be a missed expanded allele, so repeat primed PCR or Southern blot analysis is needed.
• Somatic mosaicism and instability in different tissues: the size in blood may differ from that in muscle or brain.
• Interruptions in the repeat (for example, AGG in Fragile X, CAA in Huntington disease) change stability and sometimes disease risk, and sequencing of the repeat may be needed.
• Intermediate and reduced penetrance alleles give uncertain predictions for the individual.
• Repeat size does not give a precise prediction of age at onset or the course.
• Predictive testing needs a structured counselling process, and results can have major social, psychological, insurance and employment effects.
• Testing a person tests the relatives in effect (for example, a positive parent), so confidentiality and family communication are discussed.

Special groups.

• Children: testing for adult onset diseases is deferred, and a symptomatic child is tested to confirm the diagnosis.
• At risk adults: predictive testing is voluntary, with a support person, and exclusion testing is offered in prenatal settings where the parent does not want to know their own status.
• Pregnant women and couples at risk: prenatal diagnosis or preimplantation genetic testing are options.
• Women with a Fragile X premutation: screening for ovarian insufficiency and counselling about fertility.
• Elderly carriers: neurological assessment for FXTAS and related conditions.
• Regions: access to testing is limited, and diagnosis often rests on clinical and family findings. Fragile X is under diagnosed in people with intellectual disability. Huntington disease and spinocerebellar ataxias occur in all populations, and stigma, discrimination and lack of services affect families. Low cost PCR tests in regional laboratories improve access.

Follow up tests.

• Methylation analysis and Southern blot analysis for Fragile X.
• Triplet primed PCR and sequencing of repeat interruptions.
• Testing of relatives, with counselling.
• Prenatal diagnosis, and preimplantation genetic testing.
• Baseline specialist assessments: neurology, cardiology (myotonic dystrophy, Friedreich ataxia), ophthalmology (cataract), endocrinology (diabetes in Friedreich ataxia) and psychiatry.
• Neuroimaging and neuropsychological testing.
• Genetic counselling, and support through patient organisations.