The sequencing data generated in our laboratory is analysed by our bioinformatic pipeline, integrating state-of-the art algorithms and industry-standard software solutions. We use also JSI medical systems software for sequencing data analysis. JSI medical systems is a certified system offering sophisticated bioinformatic software solutions covering a wide field of sequencing techniques.
Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results.
Every pathogenic or probably pathogenic variant is confirmed by the Sanger sequencing method. Sanger sequencing is also used occasionally with other variants reported in the statement. In the case of variant of uncertain significance (VUS) we do not recommend risk stratification based on the genetic finding. The analysis of detected variants was performed on the basis of the reference database of polymorphisms and international mutation databases: UMD, LOVD and ClinVar.
The consequence of variants in coding and splice regions are estimated using Alamut software. The Alamut database contains more than 28000 coding genes, non-protein coding genes and pseudogenes. This database (shared with the high throughput annotation engine for NGS data, Alamut Batch) is frequently updated. Information comes from different public databases such as NCBI, EBI, and UCSC, as well as other sources including gnomAD, ESP, Cosmic, ClinVar, or HGMD and CentoMD (for those a separate subscription from Qiagen/Biobase and Centogene respectively is required). Alamut Visual finds information about nucleotide conservation data through many vertebrates’ species, with the phastCons and phyloP scores, amino acid conservation data through orthologue alignments and information on protein domains.
Moreover, we integrate several missense variant pathogenicity prediction tools and algorithms such as SIFT, PolyPhen, AlignGVGD or MutationTaster. It also offers a window dedicated to the in silico study of variants’ effect on RNA splicing, allowing the assessment of their potential impact on splice junctions and visualization of cryptic or de novo splice sites. Impact on splicing regulation is also assessed.
At GHC Genetics our geneticists and clinicians, who together evaluate the results from the sequence analysis pipeline in the context of phenotype information provided in the requisition form, prepare the clinical report. We recommend an interpretation of the findings of this molecular genetic analysis, including subsequent oncological consultation for the patient in the context of genetic counselling for the patient.
We strive to continuously monitor current genetic literature identifying new relevant information and findings and adapting them to our diagnostics. This enables relevant novel discoveries to be rapidly translated and adopted into our ongoing diagnostics development without delay. The undertaking of such comprehensive due diligence ensures that our diagnostic panels and clinical statements are the most up-to-date on the market.
Variant classification is the corner stone of clinical interpretation and resulting patient management decisions. Minor modifications were made to increase reproducibility of the variant classification and improve the clinical validity of the report. Our experience with tens of thousands of clinical cases analysed at our laboratories enables us to further develop the industry standard.
The final step in the analysis of sequence variants is confirmation of variants classified as pathogenic or likely pathogenic using bi-directional Sanger sequencing. Variant(s) fulfilling all of the following criteria are not Sanger confirmed: 1) the variant quality score is above the internal threshold for a true positive call, 2) an unambiguous IGV in-line with the variant call and 3) previous Sanger confirmation of the same variant three times at GHC Genetics. Reported variants of uncertain significance (VUS) are confirmed with bi-directional Sanger sequencing only if the quality score is below our internally defined quality score for true positive call. Reported copy number variations with a size >10 exons are confirmed by orthogonal methods such as qPCR if the specific CNV has been seen less than three times at GHC Genetics.
Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes and classification of the variant). 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, 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 in 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, in the case of VUS, we do not recommend the use of genetic information in patient management or genetic counselling.
Our Clinical interpretation team analyses millions of variants from thousands of individuals with rare diseases. Thus, our database, and our understanding of variants and related phenotypes, is growing by leaps and bounds. Our laboratories are therefore well positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by GHC Genetics is re-classified, our laboratories will issue a follow-up statement to the original ordering health care provider at no additional cost.