Summary
A study of four laboratories’ experiences with subclassifying variants of uncertain significance (VUS) found that variants were more likely to be reclassified as benign than pathogenic. The VUS-high subclass had the highest percentage of reclassifications, but represented a smaller proportion of total VUS classifications. The study suggests that subclass-specific guidance for VUS may be beneficial.
Editors: Donna E. Hansel, MD, PhD, division head of pathology and laboratory medicine, MD Anderson Cancer Center, Houston; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Erica Reinig, MD, assistant professor and medical director of molecular diagnostics, University of Wisconsin-Madison; Marcela Riveros Angel, MD, molecular genetic pathology fellow, Department of Pathology, Oregon Health and Science University, Portland; Maedeh Mohebnasab, MD, assistant professor of pathology, University of Pittsburgh; Alicia Dillard, MD, associate clinical laboratory director, Omniseq/Labcorp, Buffalo, NY; and Richard Wong, MD, PhD, assistant professor of pathology, University of California San Diego.
Rates of reclassification when subclassifying VUS by evidence level
November 2025—Clinical genetic germline testing has vastly improved the understanding and characterization of many diseases and conditions. However, interpreting genetic test results is challenging, particularly with regard to variants of uncertain significance (VUS). The widely adopted current professional guidelines for classifying sequence variants, published by the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) in 2015, identify results as VUS when sufficient scoring criteria for benign or pathogenic calling are not met. To refine the classification of sequence variants that fall into the VUS category, some laboratories and other professional organizations developed independent criteria to divide this category into subclasses. Subclassification of the VUS category is proposed for the next version of the ACMG/AMP guidelines. The authors presented the experiences of four large laboratories that have been subclassifying VUS and evaluated the likelihood that variants from each subclass were reclassified over time. The laboratories have been applying VUS subclasses over a period of 10 years (Baylor Genetics), 13 years (Labcorp), 15 years (Mass General Brigham Laboratory for Molecular Medicine), and 14 years (Quest Diagnostics). A total of 40,828, 28,411, 44,113, and 44,113 variants, respectively, were classified by the institutions. While each laboratory developed its own criteria for subdividing VUS categories, all four labs created three subclasses—VUS high, VUS mid, and VUS low—that were similar in concept and evidence strengths. The authors found, across the labs, that each of the three VUS subclasses was primarily composed of missense and small in-frame deletion or insertion variants. The VUS-low subclass contained a substantially higher portion of synonymous (17 percent) and noncoding (intronic, untranslated region, others; 34 percent) variants compared with the VUS-mid (9.4 percent for the variants combined) or VUS-high (7.8 percent for the variants combined) subclasses. The authors found that when variants changed classification or subclassification, they were 4.3 times more likely to move down toward benign rather than up toward pathogenic. As expected, variants that started with a classification of benign or pathogenic were the most stable, with fewer than one percent moving away from these initial classifications. Among all categories, the VUS-high subclass had the highest percentage (10.2 percent) of variant reclassifications. Yet, because this category made up only four percent of current classifications across the four labs, this represented considerably fewer variants than for other VUS subclasses. A variant starting as VUS high was only 1.4 times more likely to move downward than upward, which is significantly less than when a single VUS class is considered. In contrast, variants starting as VUS mid or VUS low had significantly higher odds of moving down (6:1 and 9:1, respectively) when compared with a single VUS class. Overall, the classification of 96.7 percent of variants was unchanged during the study. The authors also found that the category of VUS low never moved to a Mendelian pathogenic/likely pathogenic classification. These data suggest that it may be permissible to consider de-emphasizing the category of VUS low on patient reports. Overall, the findings support the use of VUS subclasses and the development of subclass-specific professional guidance.
Bennett G, Karbassi I, Chen W, et al. Distinct rates of VUS reclassification are observed when subclassifying VUS by evidence level. Genet Med. 2025. doi.org/10.1016/j.gim.2025.101400
Correspondence: Dr. Heidi L. Rehm at [email protected]
Impact of germline genetic variation on clonal hematopoiesis landscape and progression to malignancy
The observation that cancer clusters within families led to early discoveries that heritable pathogenic germline variants predispose people to various cancers. Germline genetic variation is thought to influence tissue-specific mutational fitness, allowing clones that have a selective advantage to arise over time. Deep sequencing of normal tissue has shown that mutant clones progress to cancer in only a minority of people. Somatic mutations in hematopoietic progenitor cells create clones with varying fitness advantages in a phenomenon called clonal hematopoiesis (CH). In CH, as in normal tissue, a majority of these clones remain indolent for a patient’s lifetime. Recent studies have provided a preliminary understanding of how germline factors influence clonal hematopoiesis. The authors conducted a study in which they examined data from 731,835 people across six diverse cohorts to characterize the relationship between germline genetic variation on the CH mutational landscape and how germline-somatic interactions influence the risk of CH progressing to hematologic malignancy. Using sequencing data from the UK Biobank data set, the authors queried 236 cancer predisposition genes to identify carriers of pathogenic or likely pathogenic germline variants (PGVs). Eight percent of the biobank participants harbored a PGV in a gene with a dominant inheritance pattern. CHEK2 (0.9 percent) was the most commonly mutated gene with a dominant inheritance pattern, followed by ATM (0.5 percent) and BRCA2 (0.4 percent). To identify CH, the authors reanalyzed whole exome sequencing data from the biobank participants’ blood. Overall, 6.2 percent of people had CH in a hematologic malignancy driver gene. Clonal hematopoiesis was also found to be more prevalent in PGV carriers. CH was more strongly associated with germline-dominant carriers than with germline-recessive carriers. Among the top-10 most commonly mutated CH-heme genes, six were slightly enriched among germline carriers, with only DNMT3A and ASXL1 being statistically significant. The germline genes most associated with CH included genes implicated in DNA damage repair (CHEK2, ATM, TP53, and NBN), telomere maintenance (POT1, TINF2, and CTC1), RAS signaling (PTPN11 and SOS1), and the JAK–STAT pathway (MPL). Also identified as being associated with CH were ETV6, RUNX1, SAMD9L, and ABCB11. Most of these genes are known or hypothesized to be hematologic cancer predisposition genes, although ABCB11 had not previously been linked to hematologic cancer. Among the aforementioned 14 germline CH-predisposition genes, the authors identified marked heterogeneity in the strength of associations with acquired CH mutations. (For example, PGVs in RUNX1 often acquired SRSF2 CH.) CHEK2 PGVs were positively associated with CH in genes involved in DNA methylation. CH carriers with pathogenic germline variants were also found to have a higher risk of developing hematologic malignancy (hazard ratio, 1.3; 95 percent confidence interval, 1.1–1.5; P = 2.4 × 10−5) than CH carriers without PGVs. An exception to this pattern was for DDX41, which showed similar risks in the presence and absence of CH. A higher fraction of germline CHEK2 carriers (two percent) and ATM carriers (one percent) showed a 25-year absolute risk greater than five percent, compared with noncarriers (0.2 percent), of developing a myeloid malignancy. Taken together with other findings, the authors’ observations suggest that clonal hematopoiesis arises with normal aging and that certain germline genetic backdrops can influence CH fitness and cancer risk.
Liu J, Tran D, Xue L, et al. Germline genetic variation impacts clonal hematopoiesis landscape and progression to malignancy. Nat Genet. 2025;57:1872–1880.
Correspondence: Dr. Kelly L. Bolton at [email protected]