Somatic Inborn Errors of Immunity (IEI) Genetic Panel

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Justification

Nearly 500 genes underlie primary immunodeficiencies (PID) and in collaboration with key opinion leaders (KOLs), we have selected 69 of the most clinically actionable genes. Importantly, many PID patients have no pathogenic variants reported after testing by germline sequencing, including whole exome (WES) and whole genome (WGS) sequencing. Some of these cases will be due to somatic variants in the inborn errors of immunity (IEI) genes, increasingly recognized as an important cause of immunodeficiency (1). However, detecting somatic variants is challenging; WES/WGS does not sequence deeply enough to detect variants with low allele fractions. Furthermore, the relevant immunity genes mostly do not overlap with existing cancer genetic panels. Finally, the turnaround time is generally several weeks and these patients are frequently acutely ill. Thus, in collaboration with KOLs, we designed a somatic NGS genetic panel to fill this clinical testing gap: this test 1) sequences 69 IEI genes at ~5,000x mean read depth, 2) with 98.5% sensitivity for variant allele frequency (VAF) of 2% or greater and 100% sensitivity for VAF of 3% or greater, and 3) has a routine turnaround time of less than 1 week. Pathogenic somatic variants in a PID gene may cause the same disease as pathogenic germline variants, though the clinical presentation may be more variable in somatic cases. For example, as many as 20% of autoimmune lymphoproliferative disorder (ALPS) patients may have pathogenic somatic variants in the FAS gene (2). Similarly, somatic NLRP3 mutations were identified in ~70% of patients with mutation-negative NOMID/CINCA syndrome (3). Additionally, a recent and exciting finding is that some genes with no known role in PID can cause immune disease when somatically mutated; UBA1 (causing VEXAS) and TLR8 are examples of this striking phenomenon (4, 5). VEXAS is likely the most common somatic immune disease and was only “discovered” in 2020.

STAT: Inquire

NGS

Draw Tube: Purple Top

Sample Type: EDTA Whole Blood

Test Summary

Order Form

Specimen Requirements

	Sample Type	Volume Required	Minimum Volume	Stability
PREFERRED	EDTA Whole Blood	3mL	1mL	Room Temp.: 1 month Refrigerated: 1 month
ALTERNATIVE	DNA previously extracted from a CLIA lab	60ng of DNA (>3ng/uL)	-	Room Temp.: 1 month Refrigerated: 1 month

REJECTION CRITERIA	Sample contamination; sample compromised; buccal or FFPE specimens
SPECIAL INSTRUCTIONS	If the patient has received a bone marrow transplant, call the lab for consultation before ordering. Buccal swab is not an appropriate alternative specimen type for this somatic panel.

General Information

METHODOLOGY	NGS
STAT TAT	Inquire
STAT TAT Performance	-
ROUTINE TAT	< 5 days (M-F)
ALTERNATIVE NAMES	Somatic genetic errors of immunity
DESCRIPTION	69 genes have been sequenced and analyzed for this panel: ACD, BACH2, BRAF, CARD11, CARD14, CASP10, CDC42, COPA, CTLA4, DKC1, ELANE, ERBIN, FAS, GATA2, GFI1, IFIH1, IKBKG, JAK1, JAK2, JAK3, KRAS, MAP3K1, MAPK3, MAPK8, NLRC4, NLRP1, NLRP3, NLRP12, NOD2, NRAS, OAS1, PIK3CD, PIK3R1, PLCG2, POMP, PSMA3, PSMB4, PSMB8, PSMB9, PSMB10, PSMG2, PSTPIP1, PTEN, RIPK1, RTEL1, SAMD9, SAMD9L, SH3BP2, SOCS1, SRP54, SRP72, STAT1, STAT2, STAT3, STAT4, STAT5B, STING1, TERC, TERT, TINF2, TLR7, TLR8, TLR9, TNFAIP3, TNFRSF1A, TP53, TREX1, UBA1 and WAS. Machaon Diagnostics uses next-generation amplicon sequencing techniques (patent pending) to rapidly detect single nucleotide variants and small insertions/deletions (indels) previously reported as associated with inborn errors of immunity disorders. Novel, potentially pathogenic variants not previously reported may be discovered. Sanger sequencing or orthogonal deep re-sequencing is used for verification of all somatic variants, and as needed for germline variants (i.e., all indels or variants that fail to meet QC). The mean read depth is ~5000x and the limit of detection is a variant allele frequency 2% or greater. Genome Reference Consortium Human Build 38 (GRCh38) is the reference genome. Machaon Diagnostics uses an in-house database of variants from samples previously sequenced here plus variant data reported in the most up-to-date public databases and the published literature. For clarity, we do not report germline variants unless potentially novel or associated with our target diseases or disease risk modification in the literature. A minor allele frequency (MAF) of 0.01 indicates that the variant represents 1% of the alleles found in the population. All variants are available upon request.
LIMITATIONS	This test targets all exons of the selected genes, 5-bp of intronic DNA flanking the exon-intron boundary, plus several additional known variants of interest elsewhere in the genome; our sequencing covers 99.5% of the 69 targeted genes. This test would not detect pathogenic variants, if any, within promoter, deep intronic or untranslated regions (UTRs) or elsewhere in the genome that were not specifically targeted. Rarely, diagnostic errors may occur due to primer-site variants or mismapping of sequences to homologous regions. Known (i.e., not novel) somatic variants with a VAF in the 43.1-57.0% or > 95.7% ranges would look like germline heterozygous and homozygous variants, respectively. Our reports are based on the current understanding of these genes and diseases. This understanding may change over time as new studies are published. We recommend annual follow-up for more current interpretations; call the lab to request (510-839-5600 ext 143). The panel may miss some structural variants such as inversions, translocations, deletions/duplications, and medium/large insertions; these types of structural variants are rare and challenging to detect for any clinical method without prior knowledge of the specific structural variant. Large copy number variants (deletions or duplications spanning multiple exons) may be detected by this method; however, this test is not currently validated to detect CNVs. Natural chimeras (e.g., fusion of twin embryos in utero) and bone marrow transplant patients will be complicated candidates for this testing; please contact the lab for consultation before ordering. Buccal swab is not an appropriate alternative specimen type for this somatic panel. Other transplant patients (e.g., kidney, liver) and pregnant patients likely have cell-free DNA present in the blood; however, this cell-free DNA should not interfere with this testing. The clinical validation of this test did not include chimeras, transplant recipients or pregnant patients. Clonal hematopoiesis is a common aging-related phenomenon where a blood progenitor cell can contribute to a large genetically distinct subpopulation of blood cells and could be a source of essentially benign variants that appear suspicious. This effect is very age-dependent, observed in 10% of people > 65 years of age but in only 1% < 50 years of age.
NORMAL RANGE	Interpretation: Negative
ASSOCIATED TESTING	Soluble IL-2R alpha (CD25) Level, CXCL9 Level, IL-18 Level, HLH Extended Genetic Panel 3.0, Cytokine Release Syndrome (12-test) Panel
REFERENCES	Aluri J and Cooper M. (2023) Semin Immunol.67:101761. Lopez-Nevado M et al. (2021) Front Immunol.12:656356. Tanaka N et al. (2011) Arthritis Rheum.63(11):3625–32. Beck et al (2020) N Engl J Med. 2020;383(27):2628–38. Aluri et al. (2021) Blood. 2021;137(18):2450–62.
SAMPLE REPORT	Upon request
NEW YORK STATE APPROVED	NY Restricted Laboratory Permit Required

Test Codes

ORDER CODE	P3411
CPT CODE	81443
LOINC CODE	103741-5