Reporting of identified variants

In the final step, all identified variants along with their annotations have to undergo a medical validation
regarding their possible relevance to, the reported clinical symptoms and/or suspected diagnosis of the

For all NGS panels analyzed by Illumina, ION torrent or NGS454, all identified variants are classified
according to a standardized system of 7 classes, based on CentoMD® and the ACMG recommendations: Pathogenic (Class 1), likely pathogenic (Class 2), Variant of Unknown Significance /VUS (Class3), likely neutral (Class 4), neutral (Class 5), disease-associated SNPs (Class 6) and likely pathogenic according to our in-house CentoMD® database (Class7).

Classes 1-6 are assigned based on publicly available databases according to a standard protocol. Available databases for previously reported variants are checked (HGMD, LOVD, UMD etc.). If the variant has not been reported to date, classification relies on data regarding the frequency of the variant (ExAC Browser, Exome sequencing project 1000 Genomes and dbSNP), in silico predictions (SIFT, Polyphen,
MutationTaster ALAMUT program suite) and finally all available information regarding the segregation of the
identified variant in the patient’s family.

In WES analyses many more genes are analyzed including those without any known involvement in human disease. In these cases filtering of the identified variant is performed using the above workflow including all available gene specific information from databases such as Online Mendelian Inheritance in Man (OMIM), HGMD®, CentoMD® as well as scientific literature searches in PubMed. Due to the diagnostic setting we only report variants in genes with previously identified involvement in human diseases.

If requested by the customer we also provide information on variants in genes not associated with the
patient’s disease or symptoms. We report variants according to the recommendations of the ACMG (6).

For genes which might be involved in the patient’s disease we report all variants, except those classified as neutral or likely neutral. Disease associated polymorphisms are reported, depending on their population
frequency and importance for the individual disease phenotype.

Variants of suggested pathogenicity are reported and discussed in light of the clinical information provided, regarding their possible involvement in the patient’s disease. Recommendations for further necessary
diagnostic steps are provided based on the clinical information and genetic findings.

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