Rates and Classification of Variants of Uncertain Significance in Hereditary Disease Genetic Testing.

Importance: Variants of uncertain significance (VUSs) are rampant in clinical genetic testing, frustrating clinicians, patients, and laboratories because the uncertainty hinders diagnoses and clinical management. A comprehensive assessment of VUSs across many disease genes is needed to guide efforts to reduce uncertainty. Objective: To describe the sources, gene distribution, and population-level attributes of VUSs and to evaluate the impact of the different types of evidence used to reclassify them. Design, Setting, and Participants: This cohort study used germline DNA variant data from individuals referred by clinicians for diagnostic genetic testing for hereditary disorders. Participants included individuals for whom gene panel testing was conducted between September 9, 2014, and September 7, 2022. Data were analyzed from September 1, 2022, to April 1, 2023. Main Outcomes and Measures: The outcomes of interest were VUS rates (stratified by age; clinician-reported race, ethnicity, and ancestry groups; types of gene panels; and variant attributes), percentage of VUSs reclassified as benign or likely benign vs pathogenic or likely pathogenic, and enrichment of evidence types used for reclassifying VUSs. Results: The study cohort included 1 689 845 individuals ranging in age from 0 to 89 years at time of testing (median age, 50 years), with 1 203 210 (71.2%) female individuals. There were 39 150 Ashkenazi Jewish individuals (2.3%), 64 730 Asian individuals (3.8%), 126 739 Black individuals (7.5%), 5539 French Canadian individuals (0.3%), 169 714 Hispanic individuals (10.0%), 5058 Native American individuals (0.3%), 2696 Pacific Islander individuals (0.2%), 4842 Sephardic Jewish individuals (0.3%), and 974 383 White individuals (57.7%). Among all individuals tested, 692 227 (41.0%) had at least 1 VUS and 535 385 (31.7%) had only VUS results. The number of VUSs per individual increased as more genes were tested, and most VUSs were missense changes (86.6%). More VUSs were observed per sequenced gene in individuals who were not from a European White population, in middle-aged and older adults, and in individuals who underwent testing for disorders with incomplete penetrance. Of 37 699 unique VUSs that were reclassified, 30 239 (80.2%) were ultimately categorized as benign or likely benign. A mean (SD) of 30.7 (20.0) months elapsed for VUSs to be reclassified to benign or likely benign, and a mean (SD) of 22.4 (18.9) months elapsed for VUSs to be reclassified to pathogenic or likely pathogenic. Clinical evidence contributed most to reclassification. Conclusions and Relevance: This cohort study of approximately 1.6 million individuals highlighted the need for better methods for interpreting missense variants, increased availability of clinical and experimental evidence for variant classification, and more diverse representation of race, ethnicity, and ancestry groups in genomic databases. Data from this study could provide a sound basis for understanding the sources and resolution of VUSs and navigating appropriate next steps in patient care.

FH Variant Pathogenicity Promotes Purine Salvage Pathway Dependence in Kidney Cancer.

Fumarate accumulation due to loss of fumarate hydratase (FH) drives cellular transformation. Germline FH alterations lead to hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to an aggressive form of kidney cancer. There is an unmet need to classify FH variants by cancer-associated risk. We quantified catalytic efficiencies of 74 variants of uncertain significance. Over half were enzymatically inactive, which is strong evidence of pathogenicity. We next generated a panel of HLRCC cell lines expressing FH variants with a range of catalytic activities, then correlated fumarate levels with metabolic features. We found that fumarate accumulation blocks de novo purine biosynthesis, rendering FH-deficient cells reliant on purine salvage for proliferation. Genetic or pharmacologic inhibition of the purine salvage pathway reduced HLRCC tumor growth in vivo. These findings suggest the pathogenicity of patient-associated FH variants and reveal purine salvage as a targetable vulnerability in FH-deficient tumors. SIGNIFICANCE: This study functionally characterizes patient-associated FH variants with unknown significance for pathogenicity. This study also reveals nucleotide salvage pathways as a targetable feature of FH-deficient cancers, which are shown to be sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. This presents a new rapidly translatable treatment strategy for FH-deficient cancers. This article is featured in Selected Articles from This Issue, p. 1949.

Patterns of mosaicism for sequence and copy-number variants discovered through clinical deep sequencing of disease-related genes in one million individuals.

DNA variants that arise after conception can show mosaicism, varying in presence and extent among tissues. Mosaic variants have been reported in Mendelian diseases, but further investigation is necessary to broadly understand their incidence, transmission, and clinical impact. A mosaic pathogenic variant in a disease-related gene may cause an atypical phenotype in terms of severity, clinical features, or timing of disease onset. Using high-depth sequencing, we studied results from one million unrelated individuals referred for genetic testing for almost 1,900 disease-related genes. We observed 5,939 mosaic sequence or intragenic copy number variants distributed across 509 genes in nearly 5,700 individuals, constituting approximately 2% of molecular diagnoses in the cohort. Cancer-related genes had the most mosaic variants and showed age-specific enrichment, in part reflecting clonal hematopoiesis in older individuals. We also observed many mosaic variants in genes related to early-onset conditions. Additional mosaic variants were observed in genes analyzed for reproductive carrier screening or associated with dominant disorders with low penetrance, posing challenges for interpreting their clinical significance. When we controlled for the potential involvement of clonal hematopoiesis, most mosaic variants were enriched in younger individuals and were present at higher levels than in older individuals. Furthermore, individuals with mosaicism showed later disease onset or milder phenotypes than individuals with non-mosaic variants in the same genes. Collectively, the large compendium of variants, disease correlations, and age-specific results identified in this study expand our understanding of the implications of mosaic DNA variation for diagnosis and genetic counseling.

Fumarate Hydratase Variants and Their Association With Paraganglioma/Pheochromocytoma.

OBJECTIVE: To clarify the link between germline variants in fumarate hydratase (FH), hereditary leiomyomatosis and renal cell cancer (HLRCC), and paraganglioma (PGL) and pheochromocytoma (PCC) we utilize a well-annotated hereditary cancer testing database. METHODS: Records of 120,061 patients receiving germline testing were obtained. FH variants were classified into 4 categories: autosomal dominant (AD) HLRCC variants, autosomal recessive (AR) fumarase deficiency (FMRD), variants, previously reported as PGL/PCC FH variants, and variants of unknown significance (VUS) not previously associated with PGL/PCC (NPP-VUS). Rates of PGL/PCC were compared with those with negative genetic testing. RESULTS: About 1.3% of individuals carried FH variants which were more common among individuals with PGL/PCC compared to those without (3.1% vs 1.3%, P < .0001). PGL/PCC rates were higher among individuals with PGL/PCC FH variants compared to those with negative genetic testing (22.2% vs 0.9%, P < .0001). Neither AD HLRCC variants (0.3% vs 0.9%, P = .35) nor AR FMRD variants (1.4% vs 0.9%, P = .19) carried an increased prevalence of PGL/PCC. An increased prevalence of PGL/PCC was detected in those with NPP-VUS (2.0% vs 0.9%, P = .0023). CONCLUSIONS: Certain FH variants confer an increased risk of PGL/PCC, but not necessarily HLRCC. While universal screening for PGL/PCC among all individuals with FH variants does not appear warranted, it should be considered in select high-risk PGL/PCC FH variants.

Systematic use of phenotype evidence in clinical genetic testing reduces the frequency of variants of uncertain significance.

Guidelines for variant interpretation include criteria for incorporating phenotype evidence, but this evidence is inconsistently applied. Systematic approaches to using phenotype evidence are needed. We developed a method for curating disease phenotypes as highly or moderately predictive of variant pathogenicity based on the frequency of their association with disease-causing variants. To evaluate this method's accuracy, we retrospectively reviewed variants with clinical classifications that had evolved from uncertain to definitive in genes associated with curated predictive phenotypes. To demonstrate the clinical validity and utility of this approach, we compared variant classifications determined with and without predictive phenotype evidence. The curation method was accurate for 93%-98% of eligible variants. Among variants interpreted using highly predictive phenotype evidence, the percentage classified as pathogenic or likely pathogenic was 80%, compared with 46%-54% had the evidence not been used. Positive results among individuals harboring variants with highly predictive phenotype-guided interpretations would have been missed in 25%-37% of diagnostic tests and 39%-50% of carrier screens had other approaches to phenotype evidence been used. In summary, predictive phenotype evidence associated with specific curated genes can be systematically incorporated into variant interpretation to reduce uncertainty and increase the clinical utility of genetic testing.

Fumarate hydratase variant prevalence and manifestations among individuals receiving germline testing.

BACKGROUND: Germline variants in fumarate hydratase (FH) are associated with autosomal dominant (AD) hereditary leiomyomatosis and renal cell cancer (HLRCC) and autosomal recessive (AR) fumarase deficiency (FMRD). The prevalence and cancer penetrance across different FH variants remain unclear. METHODS: A database containing 120,061 records from individuals undergoing cancer germline testing was obtained. FH variants were classified into 3 categories: AD HLRCC variants, AR FMRD variants, and variants of unknown significance (VUSs). Individuals with variants from these categories were compared with those with negative genetic testing. RESULTS: FH variants were detected in 1.3% of individuals (AD HLRCC, 0.3%; AR FMRD, 0.4%; VUS, 0.6%). The rate of AD HLRCC variants discovered among reportedly asymptomatic individuals without a clear indication for HLRCC testing was 1 in 2668 (0.04%). In comparison with those with negative genetic testing, the renal cell carcinoma (RCC) prevalence was elevated with AD HLRCC variants (17.0% vs 4.5%; P < .01) and VUSs (6.4% vs 4.5%; P = .02) but not with AR FMRD variants. CONCLUSIONS: The prevalence of HLRCC discovered incidentally on germline testing is similar to recent population carrier estimates, and this suggests that this is a relatively common cancer syndrome. Compared with those with negative genetic testing, those with VUSs had an elevated risk of RCC, whereas those with AR FMRD variants did not.

Spectrum of splicing variants in disease genes and the ability of RNA analysis to reduce uncertainty in clinical interpretation.

The complexities of gene expression pose challenges for the clinical interpretation of splicing variants. To better understand splicing variants and their contribution to hereditary disease, we evaluated their prevalence, clinical classifications, and associations with diseases, inheritance, and functional characteristics in a 689,321-person clinical cohort and two large public datasets. In the clinical cohort, splicing variants represented 13% of all variants classified as pathogenic (P), likely pathogenic (LP), or variants of uncertain significance (VUSs). Most splicing variants were outside essential splice sites and were classified as VUSs. Among all individuals tested, 5.4% had a splicing VUS. If RNA analysis were to contribute supporting evidence to variant interpretation, we estimated that splicing VUSs would be reclassified in 1.7% of individuals in our cohort. This would result in a clinically significant result (i.e., P/LP) in 0.1% of individuals overall because most reclassifications would change VUSs to likely benign. In ClinVar, splicing VUSs were 4.8% of reported variants and could benefit from RNA analysis. In the Genome Aggregation Database (gnomAD), splicing variants comprised 9.4% of variants in protein-coding genes; most were rare, precluding unambiguous classification as benign. Splicing variants were depleted in genes associated with dominant inheritance and haploinsufficiency, although some genes had rare variants at essential splice sites or had common splicing variants that were most likely compatible with normal gene function. Overall, we describe the contribution of splicing variants to hereditary disease, the potential utility of RNA analysis for reclassifying splicing VUSs, and how natural variation may confound clinical interpretation of splicing variants.

Frequency of Cystic Fibrosis Transmembrane Conductance Regulator Variants in Individuals Evaluated for Primary Ciliary Dyskinesia.

OBJECTIVE: To evaluate whether cystic fibrosis transmembrane conductance regulator (CFTR) variants are more common among individuals tested for primary ciliary dyskinesia (PCD) compared with controls. STUDY DESIGN: Data were studied from 1021 individuals with commercial genetic testing for suspected PCD and 91 777 controls with genetic testing at the same company (Invitae) for symptoms/diseases unrelated to PCD or CFTR testing. The prevalence of CFTR variants was compared between controls and each of 3 groups of individuals tested for PCD (PCD-positive, -uncertain, and -negative molecular diagnosis). RESULTS: The prevalence of 1 pathogenic CFTR variant was similar among the individual groups. When combining the PCD-uncertain and PCR-negative molecular diagnosis groups, there was a higher prevalence of single pathogenic CFTR variants compared with controls (P = .03). Importantly, >1% of individuals who had negative genetic testing results for PCD had 2 pathogenic CFTR variants (8 of 723), and the incidence of cystic fibrosis (CF) (2 pathogenic variants) is roughly 1 in 3000 individuals of Caucasian ethnicity (∼0.03%). This incidence was also greater than that of 2 pathogenic CFTR variants in the control population (0.09% [84 of 91 777]; P = 9.60 × 10-16). These variants correlate with mild CFTR-related disease. CONCLUSIONS: Our results suggest that a single pathogenic CFTR variant is not likely to be a PCD-mimetic, but ongoing studies are needed in individuals in whom PCD is suspected and genetic testing results are uncertain or negative. Furthermore, CF may be misdiagnosed as PCD, reflecting phenotypic overlap. Among individuals evaluated for PCD, CF should be considered in the differential even in the CF newborn screening era.

Genetic analyses in a bonobo (Pan paniscus) with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder that may lead to sudden death and can affect humans and other primates. In 2012, the alpha male bonobo of the Milwaukee County Zoo died suddenly and histologic evaluation found features of ARVC. This study sought to discover a possible genetic cause for ARVC in this individual. We sequenced our subject's DNA to search for deleterious variants in genes involved in cardiovascular disorders. Variants found were annotated according to the human genome, following currently available classification used for human diseases. Sequencing from the DNA of an unrelated unaffected bonobo was also used for prediction of pathogenicity. Twenty-four variants of uncertain clinical significance (VUSs) but no pathogenic variants were found in the proband studied. Further familial, functional, and bonobo population studies are needed to determine if any of the VUSs or a combination of the VUSs found may be associated with the clinical findings. Future genotype-phenotype establishment will be beneficial for the appropriate care of the captive zoo bonobo population world-wide as well as conservation of the bobono species in its native habitat.

Expanded Gene Panel Use for Women With Breast Cancer: Identification and Intervention Beyond Breast Cancer Risk.

BACKGROUND: Clinicians ordering multi-gene next-generation sequencing panels for hereditary breast cancer risk have a variety of test panel options. Many panels include lesser known breast cancer genes or genes associated with other cancers. The authors hypothesized that using broader gene panels increases the identification of clinically significant findings, some relevant and others incidental to the testing indication. They examined clinician ordering patterns and compared the yield of pathogenic or likely pathogenic (P/LP) variants in non-BRCA genes of female breast cancer patients. METHODS: This study analyzed de-identified personal and family histories in 1085 breast cancer cases with P/LP multi-gene panel findings in non-BRCA cancer genes and sorted them into three groups by the panel used for testing: group A (breast cancer genes only), group B (commonly assessed cancers: breast, gynecologic, and gastrointestinal), and group C (a more expanded set of tumors). The frequency of P/LP variants in genes with established management guidelines was compared and evaluated for consistency with personal and family histories. RESULTS: This study identified 1131 P/LP variants and compared variants in clinically actionable genes for breast and non-breast cancers. Overall, 91.5% of these variants were in genes with management guidelines. Nearly 12% were unrelated to personal or family history. CONCLUSION: Broader panels were used for 85.6% of our cohort (groups B and C). Although pathogenic variants in non-BRCA genes are reportedly rare, the study found that most were in clinically actionable genes. Expanded panel testing improved the identification of hereditary cancer risk. Small, breast-limited panels may miss clinically relevant findings in genes associated with other heritable cancers.

Intermediate MCAD Deficiency Associated with a Novel Mutation of the ACADM Gene: c.1052C>T.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder that leads to a defect in fatty acid oxidation. ACADM is the only candidate gene causing MCAD deficiency. A single nucleotide change, c.985A>G, occurring at exon 11 of the ACADM gene, is the most prevalent mutation. In this study, we report a Caucasian family with multiple MCADD individuals. DNA sequence analysis of the ACADM gene performed in this family revealed that two family members showing mild MCADD symptoms share the same novel change in exon 11, c.1052C>T, resulting in a threonine-to-isoleucine change. The replacement is a nonconservative amino acid change that occurs in the C-terminal all-alpha domain of the MCAD protein. Here we report the finding of a novel missense mutation, c.1052C>T (p.Thr326Ile), in the ACADM gene. To our knowledge, c.1052C>T has not been previously reported in the literature or in any of the current databases we utilize. We hypothesize that this particular mutation in combination with p.Lys304Glu results in an intermediate clinical phenotype of MCADD.

BLM SUMOylation regulates ssDNA accumulation at stalled replication forks.

Polymerase stalling results in uncoupling of DNA polymerase and the replicative helicase, which generates single-stranded DNA (ssDNA). After stalling, RAD51 accumulates at stalled replication forks to stabilize the fork and to repair by homologous recombination (HR) double-strand breaks (DSBs) that accumulate there. We showed recently that SUMO modification of the BLM helicase is required in order for RAD51 to accumulate at stalled forks. In order to investigate how BLM SUMOylation controls RAD51 accumulation, we characterized the function of HR proteins and ssDNA-binding protein RPA in cells that stably expressed either normal BLM (BLM+) or SUMO-mutant BLM (SM-BLM). In HU-treated SM-BLM cells, mediators BRCA2 and RAD52, which normally substitute RAD51 for RPA on ssDNA, failed to accumulate normally at stalled forks; instead, excess RPA accumulated. SM-BLM cells also exhibited higher levels of HU-induced chromatin-bound RPA than BLM+ cells did. The excess RPA did not result from excessive intrinsic BLM helicase activity, because in vitro SUMOylated BLM unwound similar amounts of replication-fork substrate as unSUMOylated BLM. Nor did BLM SUMOylation inhibit binding of RPA to BLM in vitro; however, in immunoprecipitation experiments, more BLM-RPA complex formed in HU-treated SM-BLM cells, indicating that BLM SUMOylation controls the amount of BLM-RPA complex normally formed at stalled forks. Together, these results showed that BLM SUMOylation regulates the amount of ssDNA that accumulates during polymerase stalling. We conclude that BLM SUMOylation functions as a licensing mechanism that permits and regulates HR at damaged replication forks.

Two cases of localized neuroblastoma with multiple segmental chromosomal alterations and metastatic progression.

Surgery alone is curative for most children with localized MYCN-non-amplified neuroblastoma. However, 10-15% will develop recurrent loco-regional disease, and very rarely, patients will relapse metastatically. Currently, it is not possible to predict which child with localized, MYCN-non-amplified neuroblastoma will develop disseminated disease. We report two children who presented with favorable biology, localized neuroblastoma and subsequently relapsed with metastatic disease after treatment with surgery. Whole-genome DNA copy number analyses performed on the diagnostic tumors identified 15 (case 1) and 8 (case 2) segmental chromosomal alterations. Further analysis of the prognostic value of whole-genome analysis in children with localized neuroblastoma is warranted.

SUMO modification regulates BLM and RAD51 interaction at damaged replication forks.

The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased gamma-H2AX foci. Because the increased gamma-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess gamma-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.

Homologous recombination and maintenance of genome integrity: cancer and aging through the prism of human RecQ helicases.

Homologous recombination (HR) is a genetic mechanism in somatic cells that repairs DNA double-strand breaks and restores productive DNA synthesis following disruption of replication forks. Although HR is indispensable for maintaining genome integrity, it must be tightly regulated to avoid harmful outcomes. HR-associated genomic instabilities arise in three human genetic disorders, Bloom syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS), which are caused by defects in three individual proteins of the RecQ family of helicases, BLM, WRN, and RECQL4, respectively. Cells derived from persons with these syndromes display varying types of genomic instability as evidenced by the presence of different kinds of chromosomal abnormalities and different sensitivities to DNA damaging agents. Persons with these syndromes exhibit a variety of developmental defects and are predisposed to a wide range of cancers. WS and RTS are further characterized by premature aging. Recent research has shown many connections between all three proteins and the regulation of excess HR. Here, we illustrate the elaborate networks of BLM, WRN, and RECQL4 in regulating HR, and the potential mechanistic linkages to cancer and aging.

The DNA-binding domain of CaNdt80p is required to activate CDR1 involved in drug resistance in Candida albicans.

CaNdt80p, the Candida albicans homologue of the Saccharomyces cerevisiae transcription factor ScNdt80p, has been identified as a positive regulator of CDR1, which encodes an efflux pump involved in drug resistance in C. albicans. To investigate the involvement of the putative DNA-binding domain of CaNdt80p in drug resistance, chimeras of CaNdt80p and ScNdt80p were constructed. Interestingly, the DNA-binding domain of ScNdt80p could functionally complement that of CaNdt80p to activate CDR1p-lacZ in S. cerevisiae. Consistently, CaNdt80p containing a mutation in the DNA-binding domain failed to activate CDR1p-lacZ in S. cerevisiae. Furthermore, a copy of CaNDT80 with the same mutation also failed to complement the drug-sensitive phenotype caused by a null mutation in C. albicans. Thus, the DNA-binding domain of CaNdt80p is critical for its function in drug resistance in C. albicans.