Our Clinical Interpretation Practices - Next Generation Sequencing Assays
Clinical interpretation of next generation sequencing testing is complex and requires specific expertise. The below information provides an overview of Claritas Genomics’ Next-Generation Sequencing (NGS) interpretation practices for our clients. Technical information about Claritas’ NGS assays can be found here.
Clinical phenotyping and variant reporting: Claritas reports variants in selected genes based on either the gene region of interest(s) ordered, or for exome sequencing, the clinical information provided by the ordering provider/entity. For exome sequencing, the ability to accurately and efficiently report variants relevant to the patient relies heavily on the quality of the clinical information provided. Claritas works actively with the ordering providers to capture the major features of the patient's phenotype and apply that phenotype (expressed in HPO terms mapped to genes and/or custom gene lists) to the patient’s sequence data.
Use of analytic resources: Claritas clinical staff access a variety of resources in the course of reviewing and classifying variants. Claritas’ interpretation protocol is consistent with the ACMG/AMP standards and guidelines for the interpretation of sequence variants. Analytic resources relevant to a report or specific finding are documented as part of the testing process.
- Primary annotation databases: The principal databases used to filter and/or review variants are listed below. Databases are updated periodically to ensure provision of relevant information.
- In silico modeling tools: Claritas tests all variants against a set of gene variant predictive modeling tools, each of which has different characteristics. These tools are scientifically useful but are not validated for clinical sensitivity or specificity on their own. This information is incorporated into variant classification in a manner consistent with the ACMG/AMP guidelines.
- Prevalence databases: Claritas tests variants against databases which show both the frequency of the variant and the prevalence of the disease. The review of how often a variant is present with an actual disease or symptom is useful in determining the likelihood that a variant, acting by itself, may be pathogenic. As available databases do not fully account for differences in the frequency of particular variants in specific populations or ethnic groups, there is a risk of over reporting pathogenicity of variants in ethnic groups that are not well represented in the database(s).
Literature reviews: Claritas performs review of recent literature that may not yet be incorporated into the primary databases to complete classification of variants. Claritas provides a list of relevant citations for each variant of clinical significance in the result report.
Variants of uncertain significance: Often, laboratories detect DNA sequence variants which are of uncertain significance (“VUS”). Claritas includes VUSs in the result report according to ACMG guidelines. Some VUS variants are more likely to be associated with a patient's disease than others. Examples of factors that can increase the probability of a VUS being clinically relevant include: Variant is rare, de novo, predicted to be deleterious by computer models, and/or in a gene or region that is highly conserved during evolution.
Familial testing: Interpretation of the whole exome or large regions of interest is facilitated by comparing the patient sequence to family members, usually the parents. For example: if a variant is expected to manifest in a dominant manner, is highly penetrant, and is present in asymptomatic parents, it is unlikely to be the cause of their child’s clinical symptoms. The clinical staff may request a family member specimen if there is a possibility that it could clarify a variant classification and hence the interpretation of certain patient result reports.
Post-testing interpretive options: If a final report does not provide definitive diagnostic information, it is possible that additional follow-up could lead to useful interpretive information. For instance, looking for evidence of a clinical sign that would be predicted to be present, additional molecular testing (for instance, of other family members), or even experimental laboratory investigations (to test the impact of a variant on the function of a gene, if a suitable assay is available). Claritas can arrange for these options at the request of the provider or family via referral to the Interpretive Genomic Services at Boston Children's Hospital. This service has a number of capabilities available to further characterize the impact of a sequence variant on an individual patient.
Revisable reporting: Claritas reports may be reviewed and/or revised based on provider request.
Clinical update. A provider may have new information regarding a patient that expands the phenotype. If a request is made, a member of the clinical staff will review the new phenotype information with the provider to determine whether a patient’s sequence should be re-analyzed.
Classification changes. The classification of a variant may change over time as new information is gathered, either in the annotation databases or based on Claritas’ experience. A provider may request re-assessment of the classification of variants for a previously reported case.
Genes of uncertain significance: Claritas’ reports focus on genes that have been well established in the literature as causative of human disease. “Risk alleles” (variants which have been associated by a GWAS study with a modification of risk for a disease or condition) are not routinely reported. Also, genetic variation that lies within genes that have not been established to cause human disease, “genes of uncertain significance”, are not interpreted, or not reported. However, Claritas can make information about all of these types of variants (including risk alleles and variants in genes of uncertain significance) available to ordering physicians via its clinical gene browser (“NextCode”) upon request.
Confirmation of variants. Variant confirmation is an important component of NGS testing in order to ensure that results provided to patients do not include any false positive calls that could have a negative effect on patient care. For all ROI and exome-based tests, ~95% of all variants in the exome are confirmed using orthogonal NGS systems. We and others, (see Beck et al, Clinical Chemistry, 2016, PMID 26847218) have found NGS to be superior to Sanger sequencing for variant confirmation. Sanger sequencing is used to confirm the remaining phenotypically-relevant variants that are not confirmed by orthogonal NGS.
Gap-filling of gene regions. While most genes have good sequencing coverage, >97% of targeted bases are covered at >20X, some gene regions pose challenges for NGS and coverage may not always be 100%. When coverage is <100%, providers may request gap filling by Sanger sequencing, at no additional charge, when a single clinically-relevant variant is identified in a gene associated with a phenotypically relevant autosomal recessive condition.
ACMG secondary findings. The American College of Genetics and Genomics (ACMG) recommends assessment of a set of genes that are considered to have medical value for patient care. Secondary findings are pathogenic or likely pathogenic variants detected in this gene set. Claritas reports these secondary findings for clinical exome sequencing cases unless the consented patient/family chooses to “opt out”; i.e. not receive this information.