ThyroidCheck+

Test Methodology

The Thyroid Panel is designed to detect single nucleotide variants (SNVs) and small insertions and deletions in 59 genes associatedwith thyroid risk. Targeted regions for this panel include the coding exons and 10 bp intronic sequences immediate to the exon-intronboundary of each coding exon in each of these genes. Extracted patient DNA is prepared using targeted hybrid capture, assignment ofa unique index, and sequencing via Illumina sequencing by synthesis (SBS) technology. Data is aligned using human genome buildGRCh37. Variant interpretation is performed according to current American College of Medical Genetics and Genomics (ACMG)professional guidelines for the interpretation of germline sequence variants using Fabric EnterpriseTM Pipeline 6.6.15. Variantinterpretation and reporting is performed by Fabric Clinical (CLIA ID: 45D2281059 and CAP ID: 9619501). The following quality filtersare applied to all variants: quality <500, allelic balance <0.3, coverage <10x.

Genes Evaluated

APC, CHEK2, DICER1, DUOX2, DUOXA2, FOXE1, GLIS3, GNAS, HESX1, IGSF1, IRS4, IYD, KDM6A, KMT2D, NKX2-1, NKX2-5, OTX2, PAX8,POU1F1, PRKAR1A, PROP1, PTEN, RET, SLC16A2, SLC26A4, SLC26A7, SLC5A5, TBL1X, TG, THRA, THRB, TP53, TPO, TRHR, TSHB,TSHR, UBR1, WRN, ATP1A2, CACNA1A, CST3, CSTB, CTNNB1, G6PD,     GLIS3, HAMP, HFE, HRAS, KRAS, MECP2, NRAS, PIK3CA, PLCG2, PLN, SECISBP2, SLC40A1, TfR2, TGFBI, TTR

Test Limitations

This test aims to detect all clinically relevant variants within the coding regions of the genes evaluated. Pathogenic and likelypathogenic variants detected in these genes should be confirmed by orthogonal methods. Detected genetic variants classified asbenign, likely benign, or of uncertain significance are not included in this report. Homopolymer regions and regions outside of thecoding regions cannot be captured by the standard NGS target enrichment protocols. At this time, the assay does not detect largedeletions and duplications. This analysis also cannot detect pathogenic variants within regions which were not analyzed (e.g., introns,promoter and enhancer regions, long repeat regions, and mitochondrial sequence). This assay is not designed to detect mosaicismand is not designed to detect complex gene rearrangements or genomic aneuploidy events. It is important to understand that theremay be variants in these genes undetectable using current technology. Additionally, there may be genes associated with thyroidpathology whose clinical association has not yet been definitively established. The test may therefore not detect all variantsassociated with thyroid pathology. The interpretation of variants is based on our current understanding of the genes in this panel andis based on current ACMG professional guidelines for the interpretation of germline sequence variants. Interpretations may changeover time as more information about the genes in this panel becomes available. Qualified health care providers should be aware thatfuture reclassifications of genetic variants can occur as ACMG guidelines are updated. Factors influencing the quantity and quality ofextracted DNA include, but are not limited to, collection technique, the amount of buccal epithelial cells obtained, the patient’s oralhygiene, and the presence of dietary or microbial sources of nucleic acids and nucleases, as well as other interfering substances andmatrix-dependent influences. PCR inhibitors, extraneous DNA, and nucleic acid degrading enzymes may adversely affect assayresults.

Regulatory Disclosures

This laboratory developed test (LDT) was developed and its performance characteristics were determined by PreCheck HealthServices, Inc. This test was performed at PreCheck Health Services, Inc. (CLIA ID: 10D2210020 and CAP ID: 9101993) that is certifiedunder the Clinical Laboratory Improvement Amendments of 1988 (CLIA) as qualified to perform high complexity testing. This assayhas not been cleared or approved by the U.S. Food and Drug Administration (FDA). Clearance or approval by the FDA is not requiredfor the clinical use of this analytically and clinically validated laboratory developed test. This assay has been developed for clinicalpurposes and it should not be regarded as investigational or for research.

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^{All NGS panels have a turnaround time of 10-14 days for results.}

^{Each panel is designed to detect single nucleotide variants (SNVs) and small insertions and deletions with gene specific limitations.Targeted regions include the coding exons and 10 bp intronic sequences immediate to the exon-intron boundary of each coding exonin each of these genes.}