- Genovese G.
- et al.
GENERAL POINTS TO CONSIDER
- •Detection of aberrant clones in genetic testing has now been well described in the literature.6.,
Li–Fraumeni syndrome: not a straightforward diagnosis anymore-the interpretation of pathogenic variants of low allele frequency and the differences between germline PVs, mosaicism, and clonal hematopoiesis.Breast Cancer Res. 2019; 217.,
- Batalini F.
- et al.
- •Most commonly, these abnormal clones are derived from precursor cells of hematopoietic origin.
- •The variants identified in these cases are acquired and limited to a single cellular lineage.
- •The risk increases with older age, exposure to chemotherapy, radiation, and tobacco, and the presence of malignant or premalignant conditions (e.g., myelodysplastic syndrome, chronic lymphocytic leukemia [CLL]).
- •The association between aberrant clonal expansion (ACE) and/or CHIP and the risk for development of hematopoietic malignancy or premalignancy remains an active area of investigation.
- •The spectrum of genes most commonly impacted by ACE/CHIP has been well described,8.and some of these genes overlap significantly with those available for clinical genetic testing for hereditary cancer predisposition and other Mendelian disorders (Table 1).Table 1Most commonly altered genes in clonal hematopoiesis.
Gene Mendelian disorder (OMIM) Variant type(s) causing Mendelian disorder Mechanism of disease Short description DNMT3A Tatton–Brown–Rahman syndrome (615879) Missense/premature truncation Uncertain Autosomal dominant neurodevelopmental growth disorder TET2 None reported ASXL1 Bohring–Opitz syndrome (605039) Premature truncation Haploinsufficiency Autosomal dominant neurodevelopmental growth disorder TP53 Li–Fraumeni syndrome (151623) Missense/premature truncation Haploinsufficiency/dominant negative Hereditary cancer predisposition, often childhood onset JAK2 Thrombocythemia (614521) Missense Gain of function Autosomal dominant risks for thrombosis, cerebrovascular events and myocardial infarction SF3B1 None reported GNB1 Autosomal dominant mental retardation 42 (616973) Missense Disruption of protein–protein interactions Autosomal dominant intellectual disability disorder CBL Noonan syndrome with or without juvenile myelomonocytic leukemia (613563) Missense Haploinsufficiency/dominant negative Autosomal dominant neurodevelopmental growth disorder SRSF2 None reported PPM1D Jansen–De Vries syndrome (617450) 3’ Premature truncating (loss of nuclear localization signal) Loss of nuclear localization signal Autosomal dominant neurodevelopmental growth disorder GNAS McCune–Albright syndrome (174800)/ Pseudohypoparathyroidism (612463) Missense/premature truncation Loss of function Autosomal dominant disorder including short stature, obesity, skeletal anomalies, and hormone resistance BRCC3 None reported CREBBP Rubinstein–Taybi syndrome (180849) Premature truncation Loss of function Autosomal dominant neurodevelopmental growth disorder NRAS Noonan syndrome (613224) Missense Gain of function Autosomal dominant neurodevelopmental growth disorder RAD21 Cornelia de Lange syndrome (614701) Missense Loss of function Autosomal dominant multisystem neurodevelopmental disorder (cohesinopathy) SETDB1 None reported U2AF1 None reported SETD2 Luscan–Lumish syndrome (616831) Missense/premature truncation Loss of function Autosomal dominant neurodevelopmental growth disorder CHEK2 CHEK2-related cancer susceptibility (114480) Missense/premature truncation Loss of function Autosomal dominant hereditary cancer predisposition ATM Ataxia–telangiectasia syndrome (208900)/ ATM-related cancer susceptibility (114480) Missense/premature truncation Loss of function Autosomal recessive progressive cerebellar ataxia; autosomal dominant hereditary cancer predisposition NF1 Neurofibromatosis, type 1 (613113) Missense/premature truncation Loss of function Autosomal dominant neurodevelopmental growth disorder
- •In addition to variation at the level of the nucleotide sequence, somatic copy-number variants can be identified when genetic testing includes copy-number variant detection.
- •These may include age-related changes, such as loss or gain of the sex chromosomes in blood and bone marrow, as well as other changes such as trisomy 8, monosomy 5/deletion 5q, and monosomy 7/deletion 7q, which are common findings in aberrant hematopoietic clones and are potentially related to an underlying condition.
- •The origin (acquired or heritable) of reported genetic variants can, in some circumstances, be clarified by testing additional tissue types (e.g., cultured fibroblasts) or by identifying the variant in other family members (transmission testing).
- •Laboratories should consider recommending and/or offering these supplementary tests when a reported variant is suspicious for an acquired origin during genetic testing for hereditary disorders.
POINTS TO CONSIDER FOR TESTING LABORATORIES
- •Blood is the most common sample type accepted for germline genetic testing, and the vast majority of DNA isolated from blood derives from peripheral white blood cells (of hematopoietic origin).
- •DNA isolated from saliva and/or buccal swab samples can also be predominantly of hematopoietic origin, when isolated using standard methodologies, and the proportion of hematopoietic cell contribution varies widely, from 10% to 96% in pediatric and adult populations. This wide range may depend, in part, on the effectiveness of the user to collect a sufficient component of buccal epithelial cells.9.,
Chimerism in DNA of buccal swabs from recipients after allogeneic hematopoietic stem cell transplantations: implications for forensic DNA testing.Int. J. Legal Med. 2013; 127: 49-5410.,11.,
- Berger B.
- Parson R.
- Clausen J.
- Berger C.
- Nachbaur D.
- Parson W.
Quantitation of the cellular content of saliva and buccal swab samples.Sci. Rep. 2018; 812.
- Theda C.
- Hwang S.H.
- Czajko A.
- Loke Y.J.
- Leong P.
- Craig J.M.
- Thiede C.
- Prange-Krex G.
- Freiberg-Richter J.
- Bornhauser M.
- Ehninger G.
- •Other sample types including cultured fibroblasts, typically derived from a skin biopsy, or other available nonblood tissues should be considered to limit the risk of detecting aberrant hematopoietic clones or to follow-up a result based on DNA isolated from blood or saliva.
- •DNA derived from some tissue types, such as lymph node, spleen, or bone marrow, are also of hematopoietic origin and are not distinct from blood related findings.
- •Other solid tissue (fresh-frozen or formalin-fixed paraffin embedded) specimens may also have significant lymphocytic infiltrate present and extracted DNA may have an unexpectedly high lymphocytic origin.
- •Additionally, genetic testing performed on solid tissue may be subject to other somatic contamination (“field effects”) from adjacent malignant tissue.
- •Nontraditional DNA sources such as hair follicles and fingernails may represent alternative noninvasive sources of DNA that could be used for germline confirmation. However, testing options may be limited by the quantity of available DNA from these sources.10.,
- •Laboratories should routinely ask for clinical information as part of their test requisition, including any personal history of a hematologic condition.
- •Blood, saliva, lymph nodes, and other samples of hematologic or lymphatic system origin are not likely to be acceptable specimen types in cases where there is a history of active or overt hematologic malignancy.
- •Where no other reasonable sample type is available, a laboratory director could use their discretion to accept such samples, provided the ordering clinician consents to the potential risks of identifying a variant of indeterminate origin (somatic vs. germline).
- •Note that CLL is one of the most underappreciated diagnoses known to interfere with germline genetic testing, as active treatment is often deferred, and these samples should be accepted with caution and the consent of the ordering clinician.
- •Laboratories should routinely ask about a history of allogeneic bone marrow, peripheral stem cell, or other transplant, as these pose a substantial risk to the integrity of the sample and validity of the data as it relates to an individual’s germline DNA.
- •Laboratories should have a policy concerning the acceptability of specimens submitted following a blood transfusion.
- •In studies of cytogenetic testing following transfusion of irradiated, leukodepleted, packed red blood cells, no donor chromosomes were found in downstream analysis, suggesting that there is limited evidence to support delaying genetic testing following transfusion.15.,
- •The phenomenon of transfusion-associated microchimerism has been previously described in the setting of posttraumatic patients; however, a recent study that used single-nucleotide polymorphism (SNP) genotyping to quantify donor alleles suggests that with more modern methods for leukodepletion, this complication is rare.
- •It has been suggested that routine review of past or present test results, such as a complete blood count, peripheral smear, and flow cytometry, may help identify previously affected individuals at risk for the presence of clonal expansion, but there are limited data to support this hypothesis, and therefore the utility of such review is limited and may be cost prohibitive.
- •Additional assessment of such hematologic indices and/or laboratory review of such lab test results may not obviate the risk of aberrant clonal expansion.
- •Furthermore, these assays require specific clinical expertise for interpretation and should be reviewed by trained personnel. Such individuals may not work within a cytogenetic or molecular genetic testing laboratory.
- •At this time, clinical history remains the best predictor; often germline genetic testing results are the first indicator of clonal expansion in the blood.
Test performance and data analysis
- •NGS-based assays are exquisitely sensitive and can often identify allelic fractions as low as 5%.18.
Current practices and guidelines for clinical next-generation sequencing oncology testing.Cancer Biol. Med. 2016; 13: 3-11
- Strom S.P.
- •Heterozygous germline variants are expected to be present in one of two autosomal copies, with a normal distribution of allelic fractions centered on 50%.
- •However, there are both technical and biological reasons why these may be skewed at the time of testing, even for true germline variants.
- •In contrast, acquired variants could be present at any proportion (0–100%), but are most commonly recognized at lower levels in the absence of overt hematologic malignancy.
- •Therefore, a lower allelic fraction can be suggestive of a variant being acquired, and somatic in origin, but does not provide conclusive data.
- •In cases of a reduced allelic fraction, there also remains the possibility of postzygotic mosaicism, i.e., a variant that was acquired early on in embryonic development and is therefore present in multiple, but not all, cellular lineages, resulting in the reduced allelic fraction. This may include the germ cells in cases of gonadosomatic mosaicism. The implications for familial risk and genetic diagnosis are substantially different in these cases compared with clonal expansion of exclusively hematopoietic origin.
- •Laboratories who perform NGS-based genetic testing assays, as well as other methodologies sensitive to the detection of low-level genetic variation, should be aware of the possibility that some of the DNA variants detected may not be of germline origin, regardless of sample type.
- •Laboratories should develop quality metrics or thresholds to alert them to the presence of an acquired variant of somatic origin. Some examples might include:
- •VAF < 30%
- •Significant phenotype–genotype mismatch
- •ASXL1 pathogenic variant in an elderly male with cardiovascular disease.
- •TP53 likely pathogenic variant in a healthy adult without a personal or family history of cancer.
- •Among other factors, penetrance and age of onset should be considered when performing this comparison.
- •Consideration of the risk of postzygotic mosaicism, when known for a genetic syndrome
- •Mosaic or segmental neurofibromatosis type I (NF1) is relatively common, observed in up to 10% of cases,
- •Laboratories should consider validating methods of DNA extraction from additional tissue types, outside of blood/saliva/buccal, to facilitate further discernment of the origin of reported variants.
- •Laboratory resources, such as population databases (e.g., gnomAD) used for filtering by variant allele frequency, are also likely to harbor acquired variants.
- •The risk is that the presence of acquired variants can artificially inflate allele frequencies and lead to filtering and/or misclassification of variants as benign or likely benign, based on recommended thresholds (BA1, BS1, BS2).20.,
Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet. Med. 2015; 17: 405-424
- Richards S.
- et al.
- •Unexpected skewing in the distribution of the age of the individuals with the variant may help to flag these variants (i.e., if a variant has only been observed in older individuals that may help identify it as a potential somatic variant).
Variant interpretation and reporting
- •Prior to reporting, each DNA variant is assessed for pathogenicity and clinical significance.
- •This process includes a review of genotype–phenotype correlation.
- •Discordance should alert the laboratory to the possible presence of an acquired or somatic variant, and a potential challenge to a valid molecular diagnosis.
- •This analysis may be confounded due to concerns of reduced penetrance, de novo events, incidence of postzygotic mosaicism, etc.
- •When a variant detected through germline genetic testing is suspected to be acquired, rather than of germline origin, this should be communicated to the ordering provider on the final report. In addition, laboratories should:
- •Avoid the use of the term de novo in reporting these variants, as acquired or somatic variants are, by definition, absent from biologic parents. De novo variants are also typically associated with a risk, albeit low, of recurrence in future pregnancies, which would not be true for a somatic variant that is confined to cells of the hematopoietic lineage.
- •Rates of gonadosomatic mosaicism remain largely unknown but might be extrapolated from reports in trio exome sequencing where a limited number of variant reads have been identified in an unaffected parent of a heterozygous child.
- •Consider reanalysis with an orthogonal methodology to reduce the risk of a result due to technical reasons.
- •Recommend appropriate follow-up testing on the test report, which may include the following:
- •Acceptance of ancillary tissues to discern germline status (labs could consider including these tests as part of their germline genetic test offerings to help clarify results).
- •Site-specific testing of offspring, or other first-degree relatives, for evidence of variant transmission, not for risk assessment (cascade testing).
- •The risks related to the misapplication of a molecular diagnosis in the setting of somatic variation vary according to the gene/condition, and related management and laboratory policies should reflect the level of risk.
- •Clinical laboratories could prioritize a list of genes/conditions at risk for acquired variant detection where there is a major risk of clinical impact.
Clinical interpretation and diagnosis
- •Laboratory genetic and genomic testing has an important role in patient care and is intended to support but not replace a clinical genetic evaluation and diagnosis.
- •When genetic testing results appear inconsistent with the clinical phenotype, acquired variant detection may be an explanation, particularly in older individuals.
- •Table 1 highlights the genes where this is most likely to occur.
- •When somatic or acquired origin is suspected or confirmed, clinical management may be adjusted appropriately to avoid unnecessary treatment or intervention or further evaluate the source of the variant.
- •Additional clinical investigations (lab testing, imaging, specialty referral, etc.) may be warranted to further investigate a suspicious finding and differentiate age-related changes from a possible underlying malignancy or other condition.
ILLUSTRATIVE CASE EXAMPLES
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- Age-related mutations associated with clonal hematopoietic expansion and malignancies.Nat. Med. 2014; 20: 1472-1478
- Somatic TP53 variants frequently confound germ-line testing results.Genet. Med. 2018; 20: 809-816
- The mutational constraint spectrum quantified from variation in 141,456 humans.Nature. 2020; 581: 434-443
- Pathogenic ASXL1 somatic variants in reference databases complicate germline variant interpretation for Bohring–Opitz syndrome.Hum. Mutat. 2017; 38: 517-523
- Li–Fraumeni syndrome: not a straightforward diagnosis anymore-the interpretation of pathogenic variants of low allele frequency and the differences between germline PVs, mosaicism, and clonal hematopoiesis.Breast Cancer Res. 2019; 21
- Detection of somatic variants in peripheral blood lymphocytes using a next generation sequencing multigene pan cancer panel.Cancer Genet. 2017; 211: 5-8
- Prevalence and characteristics of likely-somatic variants in cancer susceptibility genes among individuals who had hereditary pan-cancer panel testing.Cancer Genet. 2019; 235-236: 31-38
- Chimerism in DNA of buccal swabs from recipients after allogeneic hematopoietic stem cell transplantations: implications for forensic DNA testing.Int. J. Legal Med. 2013; 127: 49-54
- Hair follicle: a reliable source of recipient origin after allogeneic hematopoietic stem cell transplantation.Bone Marrow Transplant. 2007; 40: 871-874
- Quantitation of the cellular content of saliva and buccal swab samples.Sci. Rep. 2018; 8
- Buccal swabs but not mouthwash samples can be used to obtain pretransplant DNA fingerprints from recipients of allogeneic bone marrow transplants.Bone Marrow Transplant. 2000; 25: 575-577
- Clinical implications and utility of field cancerization.Cancer Cell Int. 2007; 7
- Human nail clippings as a source of DNA for genetic studies.Open J. Epidemiol. 2015; 5: 41-50
- Fluorescence in situ hybridization and chromosome studies after transfusion in newborns: is a waiting period necessary?.Genet. Med. 2005; 7: 54-57
- Packed red cell transfusion does not compromise chromosome analysis in newborns.Genet. Med. 2001; 3: 314-317
- Lack of persistent microchimerism in contemporary transfused trauma patients.Transfusion. 2019; 59: 3329-3336
- Current practices and guidelines for clinical next-generation sequencing oncology testing.Cancer Biol. Med. 2016; 13: 3-11
- Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1).Hum. Mutat. 2011; 32: 213-219
- Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet. Med. 2015; 17: 405-424
- Updated recommendation for the benign stand-alone ACMG/AMP criterion.Hum. Mutat. 2018; 39: 1525-1530
- Clinical whole-exome sequencing for the diagnosis of mendelian disorders.N. Engl. J. Med. 2013; 369: 1502-1511
- Care of adults with neurofibromatosis type 1: a clinical practice resource of the American College of Medical Genetics and Genomics (ACMG).Genet. Med. 2018; 20: 671-682
- Clinical management of patients with ASXL1 mutations and Bohring–Opitz syndrome, emphasizing the need for Wilms tumor surveillance.Am. J. Med. Genet. A. 2015; 167A: 2122-2131
- De novo nonsense mutations in ASXL1 cause Bohring–Opitz syndrome.Nat. Genet. 2011; 43: 729-731
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