Referral for molecular genetic analysis is to:
- confirm or support a clinical diagnosis in an affected individual
- determine carrier status and / or the risk of developing symptoms in a relative of an affected individual
Variant screening for inherited cardiac and respiratory conditions
Next-generation sequencing including the analysis of copy number variants (CNVs)
Next-generation sequencing assays sequence genes associated with a range of inherited cardiac and respiratory conditions. Our 'Tests Available' page has more details of the genes and sub-panels included. All panels use targeted gene capture and sequencing is undertaken on an Illumina MiSeq or NextSeq. Sequencing data is generated on many genes; but comprehensive bioinformatic analysis, including copy number variant analysis, clinical interpretation and variant confirmation will only be reported on genes of clinical relevance to the disease category requested.
After sequencing, data goes through our state-of-the-art bioinformatics pipelines, run by highly-trained bioinformaticians. Sequencing reads are mapped to [build GRCh37 of] the human genome with the Burrows-Wheeler Aligner (BWA). Specific versions of genetic variants, called with the Genome Analysis Toolkit (GATK) HaplotypeCaller, are detailed on patient reports. Target regions are defined as coding regions ± 15bp to include splice sites.
As well as detecting single-nucleotide variants and small indels, we also identify copy-number variants (CNVs) with high sensitivity and specificity. Comprehensive variant calling is done by deep sequencing of the target regions, complemented by Sanger sequencing if necessary. All bases in exons of core genes are guaranteed to have a minimum of 20x coverage. Likely pathogenic and pathogenic variants are confirmed by Sanger sequencing, MLPA or ddPCR.
We provide a comprehensive scientific interpretation of variants found in the genes relevant to the indication. Variant annotation and assessment are carried out with public and in-house databases, to accurately assess the likelihood of pathogenicity. On completion of the analysis, a report incorporating clinical interpretation of the variants identified is given.
Sensitivity / specificity / limitations
Analytical sensitivity has been estimated to be more than 99.9% for bases covered to a minimum read depth of 20x. Analytical sensitivity for insertions and deletions may be lower in intronic poly-mononucleotide repeat regions. Specificity and precision are verified to be 100%.
Targeted variant analysis and familial testing
If a pathogenic or likely pathogenic variant is detected in a family, affected and unaffected relatives of the proband may wish to get tested for the variant. For this, we use Sanger sequencing/MLPA/ddPCR. We may also do family studies to assist with variant interpretation. For example, determining if a variant is inherited, has arisen de novo, or if the variant co-segregates with disease in a family.
All requests for familial testing must have a copy of the proband’s report, or their full details if tested in our laboratory. It is the responsibility of the referring clinician to provide this.
For samples received from family members, where the proband was tested elsewhere, optimal DNA is sent to the relevant testing lab. If this is not possible, we can carry out testing in our laboratory. Predictive testing (for unaffected relatives) should only be requested via a Clinical Geneticist or another referring clinician in association with a Clinical Geneticist.
Pathogenic and likely pathogenic SNVs and small indels detected using NGS are confirmed with Sanger sequencing. Cascade testing for these variants in family members is also available using this method.
Analytical sensitivity of this method for both single nucleotide substitutions and insertion/deletion variants is estimated to be >99%. But, non-amplification of an allele (ie.“allelic dropout”) due to unknown DNA variation within the primer binding site cannot be excluded.
Digital Droplet PCR (ddPCR) and Multiplex Ligation-dependent Probe Amplification (MLPA)
Copy number changes (CNVs) detected using NGS are confirmed, where possible, using MLPA or ddPCR. Cascade testing for these variants is also available using these methods.
Cystic fibrosis (CF)
Testing for 36 common CFTR variants is carried out using the Devyser CFTR Core kit (link). If two CF-causing variants are not detected, sequencing of the entire CFTR gene and CNV analysis is available as part of the inherited respiratory condition (Respigene) NGS panel.
Sample identity testing
Sample contamination (including Maternal Cell Contamination) detection and familial relationship confirmation can be undertaken using the PowerPlex15 kit, upon request.
We follow ACGS Genetic Laboratory Reporting Time Targets:
- Predictive testing – 14 calendar days
- Familial variant testing (non-predictive), CF common mutation screening – 28 calendar days
- Next-generation sequencing panels – 84 calendar days.
If unexpected delays occur during the issuing of results, we will keep service users informed of revised turnaround times.
Clinical genetics and genomics laboratory
Ground floor (level 2), Sydney wing, Royal Brompton Hospital, Sydney Street, London, SW3 6NP
Molecular genetic testing request and consent form (PDF, 384KB)