Factor VIII (Hemophilia A) Genetic Analysis

• Room temperature: Blood: 5 days; Swab: 60 days; DNA: 30 days

• Refrigerated: Blood: 5 days; Swab: 60 days; DNA: 30 days

• Frozen: Blood: Do not freeze; Swab: 60 days; DNA: Indefinitely

This assay will not consistently detect germline mosaicism below 50% or rule out the presence of large chromosomal aberrations, including rearrangements that do not change copy number of genomic regions. This assay will not detect inversions other than the recurrent inversions in introns 1 and 22 of the F8 gene. The assay does not detect repeat expansions. Possible intergenic variant interactions are not commented on. False positive or false negative results may occur for reasons that include: insufficient information available about rare genetic variants, sex chromosome abnormalities, pseudogene interference, homologous regions, blood transfusions, bone marrow transplantation, somatic or tissue-specific mosaicism, mislabeled samples or erroneous representation of family relationships. Variants that do not alter an amino acid composition of a protein may be difficult to assess for pathogenicity since they may produce abnormalities in structures not assessed by conventional analysis paradigms, e.g., mRNA expression and processing.¹ Interpretation of the clinical significance of gene variations is limited by information about the variant that is available at the time of reporting and by the quality and quantity of clinical information provided with the sample. As the understanding of human genetic diversity improves, the interpretation of the clinical significance of variants may change.

This test was developed and its performance characteristics determined by Labcorp. It has not been cleared or approved by the Food and Drug Administration.

Nuclear Gene Single Nucleotide Polymorphism and Small Indel Sequencing Assessment: Genomic regions of interest are selected using a custom capture reagent for target enrichment (Twist Bioscience) and sequenced via the Illumina® Novaseq 6000 Next Generation Sequencing platform. Sequencing reads are aligned with the human genome reference GRCh37/hg19 build. Regions of interest include all exons and intron/exon junctions (+/-20 nucleotides) for each gene analyzed. A minimum of 99% of bases in targeted regions are covered at >15X. Analytical sensitivity is estimated to be >99% for single nucleotide variants, >97% for insertions/deletions less than six base pairs, and >95% for insertions/deletions between six and 15 base pairs. Uncovered regions with known pathogenic variants are sequenced in a targeted manner (list based on ClinVar Database: January 20, 2020, release).

Intron 1 And Intron 22 Inversion Assessment: Regions of introns 1 and 22 of the F8 gene are amplified by long range PCR. The products are visualized by agarose gel electrophoresis and compared to control samples. Analytical sensitivity for the recurrent inversions is estimated to be >99%.

Nuclear Gene Copy Number Variant Assessment: Next Generation Sequencing data used to call SNPs and small indels are assessed with Illumina’s DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform. Genes listed in ClinVar with intragenic pathogenic deletions are padded with additional intronic probes to allow single exon resolution CNV detection (list based on ClinVar Deletion Database: January 2019 release). For other genes, large deletions (>10 exons) can be detected. The resolution of this analysis can vary depending on region-specific features. Analytical sensitivity is estimated to be >95%.

Results Interpretation: Results should be used in the context of available clinical information and should not be used as the sole basis for patient management or treatment. Genetic counseling is recommended. Variants are assessed according to ACMG criteria.² This report contains interpretation of pathogenic and likely pathogenic variants (by ACMG criteria) as well as variants of uncertain significance (VUS) with pathogenic predictions related to the clinical information provided. Variants not reported: (1) variants classified as benign or likely benign by ACMG criteria; (2) VUS with benign or likely benign predictions; (3) variants related to carrier status. We will reanalyze the data periodically at the clinician's request to allow potential reinterpretation based on new research or evidence.