Whole-Exome Sequencing

Whole exome sequencing reads the protein coding regions of nearly all genes (about 20,000 genes, around 1 to 2% of the genome, and about 30 to 60 million bases). Most disease causing variants that are known to cause single gene disorders lie in these regions, so exome sequencing is an efficient way of searching for a genetic cause without choosing a gene in advance.

DNA from blood or saliva is fragmented, the coding regions are captured by probes and sequenced, and the reads are aligned to a reference genome. Variants are filtered by frequency in the population, predicted effect, inheritance pattern and the patient's features, and then classified. Analysis of a trio (child and both parents) finds de novo and compound heterozygous variants much more easily than analysis of the child alone.

A routine result takes 4 to 8 weeks. Rapid exome sequencing in critically ill infants and children takes 3 to 10 days, and it can change management in intensive care.

The diagnostic yield is about 25 to 40% in children with developmental delay, intellectual disability or multiple congenital anomalies, about 20 to 40% in neurological and neuromuscular disease, and about 10 to 30% in foetuses with structural anomalies and a normal microarray. Re-analysis of negative cases increases the yield by about 10 to 15% over the following years, as new genes and variants are described.

Indications.

• A suspected single gene disorder with a broad, non-specific or multisystem picture, or with many possible genes.
• Unexplained developmental delay, intellectual disability, autism with other features, congenital anomalies and epilepsy.
• A critically ill infant or child with a suspected genetic disease, when a rapid diagnosis will change treatment or care decisions.
• A negative result on a gene panel, microarray or karyotype in a patient with a strong clinical suspicion.
• Prenatal diagnosis of foetal structural anomalies with normal chromosomal tests, using trio analysis.
• Undiagnosed adult disease with a strong genetic flavour (early onset, family history, multisystem).
• Sudden unexplained death with stored DNA.

Not indicated when a single gene or panel test fits the phenotype well and is cheaper and faster, or when the clinical picture does not suggest a genetic cause.

Specimen requirements.

• EDTA blood (3 to 5 mL) from the patient, and from both parents for trio analysis (preferred). Saliva is an alternative.
• A detailed clinical summary with phenotype terms, family tree, previous test results, imaging and biochemistry.
• Written consent after counselling, covering the possibility of unexpected findings, consent to report secondary findings in selected genes, data storage and sharing, and re-analysis.
• For a deceased patient, stored blood or tissue, with consent.

Normal appearances.

A negative or non-diagnostic report states that no variant explaining the phenotype was found, the proportion of coding bases covered at an adequate depth, and the limits of the method. The patient remains undiagnosed, and re-analysis is advised.

Interpretation.

• A pathogenic or likely pathogenic variant that fits the phenotype and the inheritance pattern is reported as a molecular diagnosis. Trio data show whether it arose de novo.
• A variant of uncertain significance in a gene that matches the phenotype may be reported as a possible cause that needs further evidence, such as segregation in the family or functional tests.
• Dual diagnoses: about 5% of patients have two genetic conditions that together explain a complex picture.
• Secondary findings: pathogenic variants in selected genes unrelated to the question (about 1 to 3% of people), such as cancer predisposition and cardiac genes, are reported only if the patient chose to receive them.
• Carrier findings: a heterozygous change in a recessive gene is reported only when relevant.
• Unsolved cases: the data are kept, and re-analysed after 12 to 24 months, or when the phenotype changes.
• Results are discussed in a genetic counselling visit, with advice on treatment, surveillance, recurrence risk and options for family planning.

Critical values and findings needing immediate action.

• A diagnosis in an acutely ill child that points to a treatable condition (for example, a metabolic or epilepsy disorder with a specific treatment): immediate communication to the intensive care or specialist team.
• A pathogenic variant in a gene linked to a preventable life threatening condition: surveillance and cascade testing of relatives.
• A prenatal exome result that changes counselling about the continuing pregnancy: prompt counselling by a specialist team.
• A finding of non-paternity or consanguinity that was not known: handled sensitively, as set out in the consent process.

Limitations and artefacts.

• Variants outside the coding regions (deep intronic, regulatory, promoter) are not covered, and neither are most repeat expansions, mitochondrial DNA variants in full, and balanced chromosomal rearrangements.
• Detection of copy number changes and structural variants is less sensitive than with microarray or genome sequencing.
• Coverage varies across genes, and some exons (high GC content, pseudogene regions) are poorly read.
• Low level mosaicism can be missed.
• Many variants of uncertain significance are found, and interpretation depends on the databases and the knowledge available at the time of analysis.
• Incorrect phenotype information leads to missed or wrong interpretation, so detailed clinical data are key.
• The test can reveal unexpected family relationships and carrier status, and issues of privacy and insurance need discussion before testing.
• A negative exome does not rule out a genetic cause.
• Cost, data storage and bioinformatic expertise limit access.

Special groups.

• Critically ill infants: rapid trio exome or genome sequencing can shorten the diagnostic search and affect treatment and palliative decisions.
• Children with neurodevelopmental disorders: exome sequencing is often a first line genetic test after microarray, or even before it.
• Foetuses with anomalies: trio analysis with careful counselling about uncertain results.
• Adults with undiagnosed disease: yield is lower, and a detailed phenotype helps.
• Families seeking reproductive options: a diagnosis allows carrier testing, prenatal diagnosis or preimplantation genetic testing.
• Regions: exome sequencing is accessible in few centres, mostly abroad, and costs are high. Reference data for local populations are limited, so uncertain results are more common. Consanguineous families and founder populations have high yields, since recessive disease is common. Research programmes and international collaborations give access in some places.

Follow up tests.

• Parental and family testing to check the inheritance of variants.
• Confirmation of key variants by Sanger sequencing.
• Microarray or MLPA for copy number changes, if not done before.
• Genome sequencing, long read sequencing, RNA studies or functional tests for unsolved cases.
• Enzyme, biochemical and imaging tests to support the finding.
• Cascade testing and genetic counselling for relatives.
• Specialist surveillance and treatment planning.
• Re-analysis of the data after a defined interval.