This test analyzes 51 genes associated with hereditary anemia. Both sequencing and deletion/duplication (CNV) analysis will be performed on the coding regions of all genes included (unless otherwise marked). All analysis is performed utilizing Next Generation Sequencing (NGS) technology.

Copy number variation (CNV) analysis detects deletions and duplications in the genes on the panel; in some instances, due to the size of the exons, sequence complexity, or other factors, not all CNVs may be analyzed or may be difficult to detect. Smaller CNV events may also be detected and reported, but additional follow-up testing is recommended if a smaller CNV is suspected. All variants are classified according to ACMG guidelines.

Hereditary anemia is a group of blood disorders characterized by the body's inability to produce enough healthy red blood cells (RBC) including RBC membrane structure and transport function defects, hyporegenerative anemia, RBC enzymopathies, and hyperbilirubinemia.

These conditions can lead to chronic fatigue, weakness, and shortness of breath due to insufficient oxygen delivery to tissues. Symptoms can vary from mild to severe and may include jaundice, pale skin, and delayed growth in children. The conditions can be inherited in autosomal dominant, autosomal recessive, and X-linked manners.

This panel does not include hemoglobinopathies caused by HBA1, HBA2 or HBB genes.

Sequencing is performed on genomic DNA using an Agilent targeted sequence capture method to enrich for the exome. Direct sequencing of the amplified captured regions was performed using 2X150bp reads on Illumina next generation sequencing (NGS) systems. A base is considered to have sufficient coverage at 20X and an exon is considered fully covered if all coding bases plus three nucleotides of flanking sequence on either side are covered at 20X or more. A list of these regions, if any, is available upon request.

Alignment to the human reference genome (GRCh37) is performed and annotated variants are identified in the targeted region. Variants reviewed have a minimum coverage of 8X and an alternate allele frequency of 20% or higher. Indel and single nucleotide variants (SNVs) may be confirmed by Sanger sequence analysis before reporting at director discretion. This assay cannot detect variants in regions of the exome that are not covered, such as deep intronic, promoter and enhancer regions, areas containing large numbers of tandem repeats, and variants in mitochondrial DNA.

Copy number variation (CNV) analysis detects deletions and duplications; in some instances, due to the size of the exons, sequence complexity, or other factors, not all CNVs may be analyzed or may be difficult to detect. When reported, copy number variant size is approximate. Actual breakpoint locations may lie outside of the targeted regions. CNV analysis will not detect tandem repeats, balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, and balanced insertions), methylation abnormalities, triploidy, and genomic imbalances in segmentally duplicated regions.

This assay is not designed to detect mosaicism; possible cases of mosaicism may be investigated at the discretion of the laboratory director. Primary data analysis is performed using Illumina bcl2fastq converter v2.19. Secondary analysis is performed using Illumina DRAGEN Bio-IT Platform v.3.10.8. Tertiary data analysis is performed using SnpEff v5.0 and Revvity Omics' internal ODIN v.1.01 software. CNV and absence of heterozygosity are assessed using Bionano's NxClinical v6.1 software.