Cascade (Family Variant) Testing

Cascade testing is the targeted testing of blood relatives for a genetic variant that has already been found in one member of the family (the index case). It extends the benefit of a genetic diagnosis to relatives who may share the variant, so that those at risk can be offered surveillance, prevention or treatment, and those who do not carry it can be reassured.

The test looks only for the single known variant, usually by Sanger sequencing or a targeted PCR. It is fast, cheap and very accurate. It needs a report on the index case that states the exact variant and its classification as pathogenic or likely pathogenic.

Testing starts with first degree relatives (parents, siblings and children), who have a 50% chance of carrying a variant for an autosomal dominant condition, and then extends to second degree and more distant relatives of any who carry it. For recessive conditions, siblings have a 25% chance of being affected and a 50% chance of being carriers. For X-linked conditions, risk depends on the sex of the relative and the line of descent.

Results take 1 to 3 weeks. Uptake among relatives is variable, about 30 to 50% in many programmes, so clear communication, supportive counselling and simple access to testing matter. Cascade testing is among the most effective ways of finding people who carry preventable conditions, such as familial hypercholesterolaemia, hereditary breast and ovarian cancer and Lynch syndrome.

Indications.

• Hereditary cancer syndromes: BRCA1 and BRCA2, Lynch syndrome, familial adenomatous polyposis, Li-Fraumeni syndrome, multiple endocrine neoplasia.
• Familial hypercholesterolaemia, and other inherited lipid disorders.
• Inherited heart conditions: cardiomyopathies, long QT and other channelopathies, aortopathies such as Marfan syndrome.
• Haemochromatosis, Wilson disease, and other treatable metabolic disorders.
• Haemoglobinopathies and other recessive conditions, for carrier detection before pregnancy.
• Neuromuscular and neurological conditions with a known family variant (Duchenne muscular dystrophy, spinal muscular atrophy, Huntington disease, with predictive testing procedures).
• Reproductive planning: prenatal diagnosis and preimplantation genetic testing for known variants.

Not indicated when the index variant is of uncertain significance, unless a research or segregation study has been agreed, since a result cannot direct care.

Specimen requirements.

• EDTA blood (3 to 5 mL) or saliva from the relative, with a copy of the family report or the exact variant details (gene, nucleotide change, transcript).
• Written consent after counselling about the possible results, the implications for health and for the family, privacy and insurance issues, and the right not to know.
• Information about the relationship to the index case, family tree and age.
• A positive control sample from the index case is used by the laboratory for the targeted assay.

Normal appearances.

A relative who does not carry the family variant is a true negative, and is reported as not having inherited the variant. Their risk returns to that of the general population for the condition, and their children are not at increased risk from this variant.

Interpretation.

• Positive (carrier): the relative carries the pathogenic variant, and the plan depends on the condition: surveillance, risk reducing treatment, lifestyle and medicines, and testing of their own relatives.
• Negative (true negative): applies only when the variant is confirmed as pathogenic in the index case. Routine population screening continues.
• Uninformative: if the index case has no identified variant, a negative test in a relative says little, and they are treated on the basis of family history.
• Penetrance and variability: carriers may never develop disease, or develop it at different ages and severity. A positive result gives a risk, not a certainty.
• Recessive conditions: carriers are healthy, and partner testing and reproductive options are discussed.
• Mosaicism in the index case or a de novo variant: relatives are not at risk if the variant arose de novo in the index case, though germline mosaicism is possible in a parent.
• Results are given with a personalised plan and a letter for the general practitioner.

Critical values and findings needing immediate action.

• Carriers of conditions that need treatment now (for example, familial hypercholesterolaemia in a child or adult with high LDL cholesterol, long QT syndrome with symptoms, and Marfan syndrome with aortic dilatation): start treatment and surveillance without delay.
• Carriers of hereditary cancer variants (BRCA1, BRCA2, Lynch syndrome): start surveillance from the age set for the condition, and discuss risk reducing surgery.
• A positive result in a pregnant woman: urgent counselling about prenatal options.
• A positive predictive result for an untreatable adult onset condition (Huntington disease): access to psychological support before and after disclosure.

Limitations and artefacts.

• The test depends on a correct classification of the index variant. A variant that is later reclassified as benign or uncertain changes the meaning of results in relatives.
• Different laboratories can classify the same variant differently, and a family report should state the evidence.
• Sample mix up, and unexpected family relationships (non-paternity, adoption, donor conception), can lead to misleading results and ethical problems. A second sample confirms the result when the stakes are high.
• Incomplete penetrance and wide variability limit predictions for carriers.
• Testing a person reveals the status of relatives, and issues of confidentiality, insurance, employment and family conflict need counselling.
• The duty to inform at risk relatives falls on the patient, and the clinic often supports this by a family letter. Direct contact by the clinic is limited by privacy law.
• Uptake is low where counselling, access and cost are barriers, and fear of discrimination keeps many from testing.
• Testing of minors for adult onset conditions without childhood intervention is deferred.

Special groups.

• Children: tested when an effective intervention exists in childhood (familial hypercholesterolaemia, some cardiac conditions, multiple endocrine neoplasia type 2, familial adenomatous polyposis), and not for adult onset conditions without benefit.
• Couples planning pregnancy: carrier testing and counselling for reproductive options.
• Adopted individuals and those with unknown family history: testing is considered based on clinical findings.
• Older relatives: testing depends on the benefit from surveillance and treatment.
• Families with multiple branches: a structured approach, starting with the closest relatives and moving outwards.
• Regions: cascade testing is among the most cost effective uses of genetic testing, since one confirmed family variant allows low cost testing for many relatives. This is useful for familial hypercholesterolaemia, haemoglobinopathies and hereditary cancer. Few genetic services and counselling staff are available, so clinic staff, nurses and community health workers can help with family communication. Stigma and fear of discrimination are important barriers.

Follow up tests.

• Clinical assessment of carriers (lipid profile, ECG and echocardiogram, colonoscopy, breast MRI, depending on the condition).
• Surveillance and risk reducing plans, from the age set for each condition.
• Testing of further relatives of carriers.
• Partner carrier testing for recessive conditions.
• Prenatal diagnosis and preimplantation genetic testing where relevant.
• Genetic counselling and psychological support.
• Re-contact and updating when variant classification changes.