Diagnostic yield and test sensitivity
***Rapid Genome Sequencing Service for acutely unwell children with a likely monogenic disorder (R14) is available from the 3rd October 2022 – details coming soon***
Rare monogenic paediatric onset disorders are frequently both phenotypically and genetically heterogeneous, making these disorders very difficult to diagnose.
Trio exome/genome sequencing can be used as a first line test for patients with a clinical presentation indicative of a monogenic disorder where it is likely the most cost-effective strategy or for patients where standard genetics tests have not identified a diagnosis. Please note that we do not offer singleton gene panel analysis for patients with non-syndromic intellectual delay.
Our trio “whole” exome sequencing service aims to provide a diagnosis for children with severe paediatric disorders for whom a diagnosis is required to aid clinical management, prenatal testing or pre-implantation genetic diagnosis. For lethal fetal disorders where there is insufficient fetal DNA for exome sequencing, we recommend the parental exome sequencing strategy (Ellard et al 2015 PMID 24961629; Stals et al 2018 PMID 29096039). This strategy analyses exome sequence data from parental DNA to identify potential causative heterozygous mutations, followed by co-segregation analysis by Sanger sequencing using fetal DNA. We do not provide prenatal exome sequencing where the index case is the current pregnancy and results will be used to guide prenatal management.
Please contact Karen Stals (firstname.lastname@example.org or 01392 408247 Clinical Scientist) or Dr Emma Baple (email@example.com or 01392 405749 Clinical Geneticist) to discuss patient cases or e-mail a completed Exome Request Form (see top of this page) to the laboratory.
*For users with a Mac computer: Please could the relevant Request Form be completed in Safari, or an alternative internet browser instead of Preview PDF viewer.*
From 3rd October 2022 NHS England have commissioned whole genome sequencing for acutely unwell children (R14) as part of the new National Test Directory for Rare Disease.
The trio “whole” genome sequencing test uses an inheritance based, gene agnostic approach. The advantage of sequencing the “whole” genome is that the data can be re-analysed at a later date to include newly identified disease genes. Occasionally we identify variants in a gene which is good biological candidate (but not a known disease gene) and our approach is to seek additional cases via international data sharing initiatives such as GeneMatcher or DECIPHER.
Diagnostic yield and test sensitivity for the Whole Exome Sequencing (WES) service
The likelihood of finding a genetic diagnosis by exome sequencing depends upon the patient phenotype, i.e. the prior probability that the patient has a monogenic disorder. A successful diagnosis requires that the disease gene is known, the gene is included in the exome capture and that the mutation type is detectable by the sequencing technology. Our high diagnostic yield for families tested to date is 25.8% (from 5676 cases as of 03/01/2023) with the highest diagnostic yield (48%) obtained for couples with ≥2 pregnancies affected with a lethal prenatal/neonatal disorder.
The exome sequencing service is UKAS accredited (ISO15189). We use Twist exome capture reagents and Illumina next generation sequencing. Read depth is a key determinant of variant detection sensitivity. In our “whole” exome assay (using the Twist exome capture) the average percentage of coding nucleotides of targeted RefSeq exons (https://www.ncbi.nlm.nih.gov/refseq/) with ≥20 reads is 98.8%. The sensitivity for heterozygous single nucleotide variant (SNV) detection at a read depth of 20 is estimated at 99.89% (95%CI 99.88-99.91%). Small insertions and deletions (indels up to 30bp) are more difficult to detect and depends upon both the type of indel, the size and sequence context. The sensitivity for detecting heterozygous indels at a read depth of 20 is estimated at 98.6% (95%CI 97.8-99.12%). The overall sensitivity for SNV/indel detection (based on a 3:1 ratio of SNVs:indels) at 20X read depth is predicted to be >99%. This technology also enables the detection of copy number variants (CNV) using SavvyCNV analysis software (https://github.com/rdemolgen/SavvySuite). The sensitivity for CNV detection has not been formally tested using a large set of positive controls and therefore this method cannot exclude the presence of a CNV. As of April 8th 2019, exome sequence analysis has identified CNVs in 21 index cases from a total of 365 new diagnoses (5.8%). These include six single exon deletions (see White et al. 2017 PMID 29276006 for NXN case), thirteen multi-exon deletions and two cases of a gene cluster duplication.
DNA samples required
Please send 1µg DNA extracted from a peripheral blood EDTA sample for each individual for whom exome sequencing is requested. Saliva samples are accepted in exceptional circumstances but DNA from blood is preferred because the variable amount of bacterial DNA in saliva will reduce the amount of sequence read data for the patient.
Risk of incidental findings
Our policy is to focus on identifying disease-causing variants of direct relevance to the clinical referral. The strategies we employ result in a low prior probability of identifying mutations that predispose to other rare diseases but the possibility of incidental findings cannot be excluded and may be discussed with the referring clinician on a case-by-case basis. Trio exome sequencing will reveal possible non-paternity (or non-maternity) and this result would be discussed with the referring clinician.
Whilst there is growing evidence that many patients are keen to receive information about additional, clinically actionable findings (Middleton et al Eur J Hum Genet 2015 PMID 25920556), we await the results of studies such as the 100,000 Genomes Project (www.genomicsengland.co.uk) to understand the overall risks and benefits of receiving such information. Meanwhile the variant data from exome sequencing tests will be stored long-term in order that further analysis can be undertaken in the future.
Our exome sequencing test currently takes 2-10 weeks for routine referrals and 2-3 weeks for urgent referrals from receipt of suitable samples to issue of the report(s). This includes the pre-sequencing processing, next generation sequencing, data analysis and confirmation of likely pathogenic variants. The exact timings depend on factors including sample batching, scheduling of next generation sequencing runs and any instrument breakdowns as well as the time required to obtain additional information or samples for co-segregation testing. The national target for urgent and non-urgent clinical exome sequencing is that >90% of reports are issued within 3 and 16 weeks respectively.
No diagnosis found
For trio exome analysis where likely causative variant(s) are not identified through the inheritance based approach, please let us know if there is a specific diagnosis or genetic aetiology that you suspect (if not already included on the request form) and provide a short list of genes that we can check more closely. Trio analysis will not detect a heterozygous variant inherited from an unaffected parent, so please contact us if a non-penetrant disorder is suspected to discuss options for additional analysis. If you’d like to check the read depth coverage of a gene, genes or gene panel, please use our new tool (see exome coverage data tool updated 04/02/2022).
Please note that a diagnosis might be identified at a later date as a result of the implementation of updates to our analysis pipeline.