Whole Exome Family
Whole Exome Family includes high-quality whole exome sequence analysis of an index patient and parents (trio) or other family members, coupled with whole exome deletion/duplication analysis and mitochondrial genome (mtDNA) sequence analysis. Whole Exome Family is essential tool for detecting de novo mutations and copy number variants, which underlie many of the early-onset diseases.
Whole-exome sequencing (WES) is a robust and one of the most comprehensive genetic tests to identify the disease-causing changes in a large variety of genetic disorders. In WES, protein-coding regions of all nuclear and mitochondrial genes (~20,000) of the human genome, ie, exome, are sequenced using next-generation sequencing technologies. While the exome constitutes only ~1% of the whole genome, 85% of all disease-causing mutations are located there.
WES is most suitable for individuals with:
- Complex phenotypes with multiple differential diagnoses
- Genetically heterogeneous disorders
- Suspected genetic disorders where a specific genetic test is not available
- Inconclusive previous genetic testing
Blueprint Genetics Whole Exome tests have been developed to maximize diagnostic yields, first of all, by generating high-quality and uniform sequencing data. The sequencing data are analysed using an in-house, state-of-the art bioinformatics pipeline. Furthermore, the genetic information of patients is carefully interpreted by our team of geneticists and clinicians, utilising information from the latest publications and up-to-date databases.
This test is not available for prenatal samples. Test is available in the US, LATAM, Europe, Middle East, Canada, Asia, Australia.
- NGS
Whole Exome Family includes high-quality whole exome sequence analysis of an index patient and parents (trio) or other family members, coupled with whole exome deletion/duplication analysis and mitochondrial genome (mtDNA) sequence analysis. Whole Exome Family is essential tool for detecting de novo mutations and copy number variants, which underlie many of the early-onset diseases.
Whole-exome sequencing (WES) is a robust and one of the most comprehensive genetic tests to identify the disease-causing changes in a large variety of genetic disorders. In WES, protein-coding regions of all nuclear and mitochondrial genes (~20,000) of the human genome, ie, exome, are sequenced using next-generation sequencing technologies. While the exome constitutes only ~1% of the whole genome, 85% of all disease-causing mutations are located there.
WES is most suitable for individuals with:
- Complex phenotypes with multiple differential diagnoses
- Genetically heterogeneous disorders
- Suspected genetic disorders where a specific genetic test is not available
- Inconclusive previous genetic testing
Blueprint Genetics Whole Exome tests have been developed to maximize diagnostic yields, first of all, by generating high-quality and uniform sequencing data. The sequencing data are analysed using an in-house, state-of-the art bioinformatics pipeline. Furthermore, the genetic information of patients is carefully interpreted by our team of geneticists and clinicians, utilising information from the latest publications and up-to-date databases.
This test is not available for prenatal samples. Test is available in Europe, Middle East, Canada, Asia, Australia.
For test available in USA and South America go here.
Testing to evaluate family relatedness may be performed as part of our Quality Control (QC) process. Please see the Discordant Relatedness entry on our FAQ page for more details on how we define and handle discordant relatedness when identified.
Test Strenghts and Limitations
Test strength
The strengths of this test include:
- CAP and ISO-15189 accreditations covering all operations at Blueprint Genetics including all Whole Exome Sequencing, NGS panels and confirmatory testing
- CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory
- Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance
- Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level
- Our publically available analytic validation demonstrating complete details of test performance
- ~2000 non-coding disease causing variants in Blueprint WES assay
- Our rigorous variant classification based on modified ACMG variant classification scheme
- Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data
- Our comprehensive clinical statements
Test limitations
Genes with partial, or whole gene, segmental duplications in the human genome are listed in our website (https://blueprintgenetics.com/pseudogene/) if they overlap with the UCSC pseudogene regions. The technology may have limited sensitivity to detect variants in these genes.
This test does not detect the following:
- Complex inversions
- Gene conversions
- Balanced translocations
- Repeat expansion disorders unless specifically mentioned
- Non-coding variants deeper than ±20 base pairs from exon-intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).
This test may not reliably detect the following:
- Low level mosaicism (variant with a minor allele fraction of 14.6% is detected with 90% probability)
- Stretches of mononucleotide repeats
- Indels larger than 50bp
- Single exon deletions or duplications
- Variants within pseudogene regions/duplicated segments
This test is not available for prenatal samples.
The sensitivity of this test may be reduced if DNA is extracted by a laboratory other than Blueprint Genetics.
For additional information, please refer to the Test performance section and see our Analytic Validation.
We utilize whole exome capture technology and Next-Generation Sequencing methods to obtain clinical-grade WES data, maximizing coverage of clinically relevant genes. The performance metrics listed below are from validation performed at our laboratory in Finland.
- Highly uniform sequencing depth across all protein-coding genes of the genome
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- Mean sequencing coverage on average 154x at 100M sequenced reads
- On average, 99.6% of base pairs in target region is covered at least 20x
- Target region: protein coding exons extending ±20 base pairs to intron-exon boundaries and ~2,000 intronic regions know to contain medically relevant variants
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- Highly sensitive and specific detection of single-nucleotide variants and indels (GIAB 4.2.1)
- 99.1% (99.8%*) sensitivity and >99.99% specificity for detecting single-nucleotide variants within target region
- 95.0% (94.5%*) sensitivity and >99.99% specificity for indel (≤ 50 bps) detection within target region
- Range of detected indels in validation: Deletions up to 1-245bp detected, insertions 1-200bp
- Assay performs with high precision
- Within-run precision (repeatability) 99.1%, intermediate precision (reproducibility) 99.0%
- Sensitive and specific detection of copy number variants (CNVs)
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- In validation single exon deletion (and larger) events were detected at 100% sensitivity.Segmentally duplicated genomic regions may have reduced sensitivity. The exact boundaries of the copy number aberration cannot be determined with this test
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Analytical sensitivity to detect single-nucleotide variants and indels were calculated using latest version of high-confidence benchmark data provided byGenome in a Bottle (v4.2.1).
Values indicated with * were generated using previous version (v3.3.2) of benchmark data. Version 4.2.1 is extended to included challenging medically relevant regions and other difficult to map regions. Version 4.2.1 covers 94.1% of reference (GRCh37) and v3.3.2 covers 87.8% of reference. For more information see https://doi.org/10.1016/j.xgen.2022.100128
The performance of Blueprint Genetics mtDNA Testing
Heteroplasmy detection capabilities
- SNVs: 100% sensitivity for 5% and higher heteroplasmy
- Indels: 100% sensitivity for 5% and higher heteroplasmy
- Large 500bp – 5,000kb deletions: down to 10% (at 99% sensitivity)
Coverage
- Mean sequencing depth of 9,362x with 100M sequenced reads
- 100% of base pairs covered at 1,000x
Whole exome sequencing targets all protein coding exons and ± 20 base pairs from the exon-intron boundary. In addition, the test includes Mitochondrial Genome Sequencing (mtDNA) and around 2000 selected non-coding, deep intronic disease causing variants (listed in Appendix 8 in the electronic version of the exome report).
The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and software solutions. The proprietary automated bioinformatics pipeline is streamlined to maximize sensitivity without sacrificing specificity. It enables detection of single-nucleotide and small indel variants from WES data in addition to large copy-number variants (≥1 exon level) when Plus analysis is requested. Quality control steps are included throughout to ensure the consistency, validity and accuracy of results.
- WES data are primarily analyzed for changes in genes that are known to be associated with human disease. We monitor recent literature and up-to-date databases to link variants in genes observed in patients with up-to-date information regarding the genes’ association with relevant diseases. To further aid the process of variant interpretation, observed variants are matched against a comprehensive set of databases of disease-related mutations, collected and curated in-house, and accessed from the public domain or licensed from commercial sources. We have incorporated a number of reference population databases and mutation databases such as, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico tools such as SIFT, PolyPhen and MutationTaster are used to assist with variant classification. Splicing analysis is carried out by using Alamut Visual Software (SpliceSiteFinder-like, MaxEntScan, NNSPLICE, GeneSplicer).
Through our online ordering and statement reporting system, Nucleus, the customer has access to details of the analysis, including patient specific sequencing metrics such as coverage and sequencing depth. This reflects our mission to provide fully transparent genetic diagnostics where customers have easy access to key details of the analysis.
During the analysis of the WES data, we are looking for a genetic explanation for the patient’s symptoms. Therefore, analysis and reporting focus on variants that are directly related to the patient’s phenotype. This includes known/possibly disease-causing heterozygous variants in genes associated with autosomal dominant (AD) conditions, homozygous/compound heterozygous variants in genes associated with autosomal recessive (AR) conditions, or heterozygous/hemizygous/homozygous variants associated with X-linked disorders that are consistent with all, or a portion of, the patient´s phenotype as reported to the laboratory.
We use a variant-driven approach, often referred to as a ‘genotype-first’ strategy in the literature. This approach is considered to be one of the major benefits of WES and whole genome sequencing as it means that we do not pre-filter the sequencing data against predefined set of genes that are thought to be associated with patient’s disease but instead review all identified variants in all protein coding genes complemented by non-coding genes with a known association with human disease. The genotype-first approach considers that many patients referred for WES may have 1) an atypical presentation of a relatively well-known syndrome, 2) a genetically highly heterogeneous syndrome, 3) a very rare disease with a clinical picture that has not yet been well-established or 4) the possibility of multiple diagnoses that may confound the clinical presentation.
The clinical and family history of the patient, including symptoms, age of onset and prevalence and inheritance pattern of the disease are all taken into consideration. It is therefore important that the clinical and family history information provided is as detailed and complete as possible to ensure all relevant variants are reported. Carrier status of variants in genes not related to the patient’s phenotype are not specifically assessed and are not reported.
Analysis of the WES data first focuses on genes that have an established association with genetic disorders. The genes with a known clinical association include those curated by Blueprint Genetics (BpG) and included in BpG diagnostic panels. This list is supplemented with genes included in The Clinical Genomics Database and the Developmental Disorders Genotype-Phenotype Database (DD2GP). The total number of genes that are considered clinically associated is currently in the order of 3800 although this number is constantly changing.
If analysis of variants in previously established disease genes is inconclusive, exome data are also analyzed for variants that are not located within known clinically associated genes but have properties that make them candidates to be disease-causing. These properties include: 1) de novo variants in coding regions (for probands who were whole exome sequenced with parents), 2) novel heterozygous truncating variants in genes predicted to be intolerant for loss-of-function variation based on ExAC variant data (probability of loss-of-function intolerance score pLI≥0.9) or 3) rare homozygous truncating or compound heterozygous variants, or a combination of rare truncating and rare missense variants that are predicted deleterious by multiple in silico tools. Only variants in genes with expression pattern and function considered relevant for the phenotype are included in the clinical report.
As WES covers all protein-coding genes of the genome, it enables detection of variants that are not associated with the indication for performing WES but are of medical value for patient care. These findings are known as secondary findings. Blueprint Genetics follows the ACMG SF 3.1 List for Reporting of Secondary Findings in Clinical Exome and Genome Sequencing to seek and report clinically actionable variants in genes determined by ACMG. These guidelines are followed if the patient or caregiver has opted-in for analysis and reporting of secondary findings. If parents or other family members are also participating in the WES analysis, they have the option to opt-in for analysis and reporting of secondary findings. Secondary findings for additional family members are reported in a separate clinical statement than that of the index patient and is independent of the secondary findings reported in the index patient.
Variant classification is the corner stone of clinical interpretation and the resulting patient management decisions. Our classifications follow the ACMG guideline 2015.
The final step in the analysis is orthogonal confirmation. Sequence variants classified as pathogenic, likely pathogenic and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing when they do not meet our stringent NGS quality metrics for a true positive call. Reported copy number variations with a size <10 exons are confirmed by orthogonal methods such as dPCR if the specific CNV has been seen less than three times at Blueprint Genetics.
We aim to provide customers with the most comprehensive clinical statement available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists and clinical consultants prepare the clinical statement by evaluating the identified variants in the context of the phenotypic information provided on the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.
The clinical statement features tables that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes and classification of the variant) for the sequencing and copy number variants identified. In addition, the statement includes detailed descriptions of the variant, gene and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts and detailed information about related phenotypes. We also provide links to the references used, congress abstracts and mutation databases to help our customers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.
Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations on different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification within the family. In the case of variants of uncertain significance (VUS), we do not recommend family member risk stratification based on the VUS result. Furthermore, a VUS should not be used to determine patient management or clinical decision making (Richards et al., 2015).
Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our database, and our understanding of genetic variants and related phenotypes, continues to grow. Our laboratory is therefore well positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering health care provider at no additional cost.
As WES includes all protein-coding genes of the genome, it detects variants in genes that are not related to the indication for performing WES but are of medical value for patient care. These variants are known as secondary findings. Blueprint Genetics follows the ACMG recommendations for reporting secondary findings in clinical exome and genome sequencing to seek and report clinically actionable variants in genes determined by ACMG. These guidelines are followed if the patient or caregiver has opted-in for the analysis and reporting of secondary findings. If parents or other family members are also participating in the WES analysis, they also have the option to opt-in for analysis and reporting of secondary findings. Secondary findings for additional family members are reported in a separate clinical statement than that of the index patient and are independent of the secondary findings reported in the index patient.