Whole Exome Family

Whole Exome Family includes high-quality Whole Exome sequence analysis of an index patient and parents (trio) or other family members. Trio approach in WES improves diagnostic rate by facilitating sequence variant analysis and by enabling detection of de novo mutations, which underlie many of the severe 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 genes (~20,000) of the human genome, i.e. 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

• a complex, unspecific genetic disorder with multiple differential diagnoses.
• a genetically heterogeneous disorder.
• a suspected genetic disorder where a specific genetic test is not available.
• unsuccessful 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 analyzed using in-house, state-of-the art bioinformatics pipeline. Furthermore, the genetic information of patients is carefully interpreted by our team of geneticists and clinicians, utilizing information from latest publications and up-to-date databases.

Whole exome sequencing targets all protein coding exons and ± 20 base pairs from the exon-intron boundary. In addition, the test includes >1500 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.

Variant classification is the corner stone of clinical interpretation and the resulting patient management decisions. Our classifications follow the Blueprint Genetics Variant Classification Schemes based on the ACMG guideline 2015. Minor modifications, based on our experience with tens of thousands of patient results, were made to increase reproducibility of the variant classification and improve the clinical validity of the report. Our statements include a comprehensive description of our rationale for the classification of the variant.

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 covers all protein-coding genes of the genome, it enables detection of variants that are not associated with the indication for ordering the sequencing but are of medical value for patient care. These kind of findings are called secondary or incidental findings. We follow the ACMG Recommendations for Reporting Incidental Findings in Clinical Exome and Genome Sequencing to seek and report clinically actionable mutations of specified types in 59 genes determined by ACMG, if the patient or the caregiver has opted-in for analysis and reporting of secondary findings. If parents or other family members are also subjected to WES, they also have the possibility to opt-in for analysis and reporting of secondary findings. Secondary findings are reported in a separate statement. All reported secondary findings variants are based on high-quality variant calls in NGS data but these variants do not go through Sanger confirmation, which is in-line with the ACMG policy. Secondary findings are not analyzed or reported for deceased individuals or fetal samples.