Trafficking of mitochondrial double-stranded RNA from mitochondria to the cytosol.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.

A method for measuring mitochondrial DNA copy number in pediatric populations.

The mitochondrion is a multifunctional organelle that modulates multiple systems critical for homeostasis during pathophysiological stress. Variation in mitochondrial DNA (mtDNA) copy number (mtDNAcn), a key mitochondrial change associated with chronic stress, is an emerging biomarker for disease pathology and progression. mtDNAcn can be quantified from whole blood samples using qPCR to determine the ratio of mtDNA to nuclear DNA. However, the collection of blood samples in pediatric populations, particularly in infants and young children, can be technically challenging, yield much smaller volume samples, and can be distressing for the patients and their caregivers. Therefore, we have validated a mtDNAcn assay utilizing DNA from simple buccal swabs (Isohelix SK-2S) and report here it's performance in specimens from infants (age = <12 months). Utilizing qPCR to amplify ∼200 bp regions from two mitochondrial (ND1, ND6) and two nuclear (BECN1, NEB) genes, we demonstrated absolute (100%) concordance with results from low-pass whole genome sequencing (lpWGS). We believe that this method overcomes key obstacles to measuring mtDNAcn in pediatric populations and creates the possibility for development of clinical assays to measure mitochondrial change during pathophysiological stress.

Pediatric Chordoma: A Tale of Two Genomes.

Little is known regarding the genomic alterations in chordoma, with the exception of loss of SMARCB1, a core member of the SWI/SNF complex, in poorly differentiated chordomas. A TBXT duplication and rs2305089 polymorphism, located at 6q27, are known genetic susceptibility loci. A comprehensive genomic analysis of the nuclear and mitochondrial genomes in pediatric chordoma has not yet been reported. In this study, we performed whole exome and mitochondrial DNA (mtDNA) genome sequencing on 29 chordomas from 23 pediatric patients. Findings were compared with that from whole genome sequencing datasets of 80 adult skull base chordoma patients. In the pediatric chordoma cohort, 81% percent of the somatic mtDNA mutations were observed in NADH complex genes, which is significantly enriched compared to the rest of the mtDNA genes (p=0.001). In adult chordomas, mtDNA mutations were also enriched in the NADH complex genes (p<0.0001). Furthermore, a progressive increase in heteroplasmy of non-synonymous mtDNA mutations was noted in patients with multiple tumors (p=0.0007). In the nuclear genome, rare likely germline in-frame indels in ARID1B, a member of the SWI/SNF complex located at 6q25.3, were observed in five pediatric patients (22%) and four patients in the adult cohort (5%). The frequency of rare ARID1B indels in the pediatric cohort is significantly higher than that of the adult cohort (p=0.0236, Fisher's exact test), but they were both significantly higher than that in the ethnicity-matched populations (p<5.9e-07 and p<0.0001174, respectively). Implications: germline ARID1B indels and mtDNA aberrations appear important for chordoma genesis, especially in pediatric chordoma.

Significance Associated with Phenotype Score Aids in Variant Prioritization for Exome Sequencing Analysis.

Several in silico annotation-based methods have been developed to prioritize variants in exome sequencing analysis. This study introduced a novel metric Significance Associated with Phenotypes (SAP) score, which generates a statistical score by comparing an individual's observed phenotypes against existing gene-phenotype associations. To evaluate the SAP score, a retrospective analysis was performed on 219 exomes. Among them, 82 family-based and 35 singleton exomes had at least one disease-causing variant that explained the patient's clinical features. SAP scores were calculated, and the rank of the disease-causing variant was compared with a known method, Exomiser. Using the SAP score, the known causative variant was ranked in the top 10 retained variants for 94% (77 of 82) of the family-based exomes and in first place for 73% of these cases. For singleton exomes, the SAP score analysis ranked the known pathogenic variants within the top 10 for 80% (28 of 35) of cases. The SAP score, which is independent of detected variants, demonstrates comparable performance with Exomiser, which considers both phenotype and variant-level evidence simultaneously. Among 102 cases with negative results or variants of uncertain significance, SAP score analysis revealed two cases with a potential new diagnosis based on rank. The SAP score, a phenotypic quantitative metric, can be used in conjunction with standard variant filtration and annotation to enhance variant prioritization in exome analysis.

MSeqDR Quick-Mitome (QM): Combining Phenotype-Guided Variant Interpretation and Machine Learning Classifiers to Aid Primary Mitochondrial Disease Genetic Diagnosis.

The international Mitochondrial Disease Sequence Data Resource Consortium (MSeqDR) Quick-Mitome (QM) is a web-based platform enabling automated variant interpretation of whole-exome sequencing (WES) datasets for the genetic diagnosis of primary mitochondrial diseases (PMD). Designed specifically to address the unique dual genome nature of PMD etiologies, QM includes features for both nuclear and mitochondrial DNA (mtDNA) genome analysis. QM requires VCF variant lists, HPO ID clinical phenotypes, and pedigree files for multiple-sample VCF inputs. QM maps phenotypes to HPO terms before analysis. QM analysis requires 2 to 20 min for 100,000 variants on an 8-vCPU AWS server using Exomiser's "PASS_ONLY" mode for nuclear variants. QM ranks variants based on allele frequency, phenotype-gene association, functional impact, and inheritance mode. Variants are further annotated with multiple data sources such as OMIM, ClinVar, dbNSFP, gnoMAD, MITOMAP, and MSeqDR. In addition to standard Exomiser results, QM generates an Analysis Report and QM Integrated Report with add-on mtDNA-specific analyses, including haplogroup prediction with Phy-Mer, heteroplasmy calculation, and mvTool annotations. We developed the Mitochondrial Disease Variant (MDV) classifier using XGBoost to predict variant pathogenicity for PMD. The MDV classifier was trained on >120 features and performance benchmarking showed that it correctly classified >98% of nuclear gene variants as being pathogenic or benign, and predicted PMD-causing variants with 94% precision. The MSeqDR QM server is an open-access resource for phenotype-driven dual-genome analyses for PMD diagnosis by the global mitochondrial disease community. It is publicly available for non-commercial, non-clinical research use at https://mseqdr.org/quickmitome.php. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Standardizing clinical phenotypes into human phenotype ontology (HPO) terms as the phenotype input for Quick-Mitome (QM) Basic Protocol 2: Prepare the pedigree input for multiple-sample VCF Basic Protocol 3: Quick-Mitome (QM) analysis Basic Protocol 4: Reviewing and understanding the QM Integrated Report and Analysis Report.

An Exome Capture-Based RNA-Sequencing Assay for Genome-Wide Identification and Prioritization of Clinically Important Fusions in Pediatric Tumors.

This study reports the development of an exome capture-based RNA-sequencing assay to detect recurring and novel fusions in hematologic, solid, and central nervous system tumors. The assay used Twist Comprehensive Exome capture with either fresh or formalin-fixed samples and a bioinformatic platform that provides fusion detection, prioritization, and downstream curation. A minimum of 50 million uniquely mapped reads, a consensus read alignment/fusion calling approach using four callers (Arriba, FusionCatcher, STAR-Fusion, and Dragen), and custom software were used to integrate, annotate, and rank the candidate fusion calls. In an evaluation of 50 samples, the number of calls varied substantially by caller, from a mean of 24.8 with STAR-Fusion to 259.6 with FusionCatcher; only 1.1% of calls were made by all four callers. Therefore a filtering and ranking algorithm was developed based on multiple criteria, including number of supporting reads, calling consensus, genes involved, and cross-reference against databases of known cancer-associated or likely false-positive fusions. This approach was highly effective in pinpointing known clinically relevant fusions, ranking them first in 47 of 50 samples (94%). Detection of pathogenic gene fusions in three diagnostically challenging cases highlights the importance of a genome-wide and nontargeted method for fusion detection in pediatric cancer.

Low-pass whole-genome and targeted sequencing of cell-free DNA from cerebrospinal fluid in pediatric patients with central nervous system tumors.

Background: Central nervous system tumors are the most common pediatric solid tumors and the most frequent cause of cancer-related morbidity in childhood. Significant advances in understanding the molecular features of these tumors have facilitated the development of liquid biopsy assays that may aid in diagnosis and monitoring response to therapy. In this report, we describe our comprehensive liquid biopsy platform for detection of genome-wide copy number aberrations, sequence variants, and gene fusions using cerebrospinal fluid (CSF) from pediatric patients with brain, spinal cord, and peripheral nervous system tumors. Methods: Cell-free DNA was isolated from the CSF from 55 patients, including 47 patients with tumors and 8 controls. Results: Abnormalities in cell-free DNA were detected in 24 (51%) patients including 11 with copy number alterations, 9 with sequence variants, and 7 with KIAA1549::BRAF fusions. Positive findings were obtained in patients spanning histologic subtypes, tumor grades, and anatomic locations. Conclusions: This study demonstrates the feasibility of employing this platform in routine clinical care in upfront diagnostic and monitoring settings. Future studies are required to determine the utility of this approach for assessing response to therapy and long-term surveillance.

Resilience to autosomal dominant Alzheimer's disease in a Reelin-COLBOS heterozygous man.

We characterized the world's second case with ascertained extreme resilience to autosomal dominant Alzheimer's disease (ADAD). Side-by-side comparisons of this male case and the previously reported female case with ADAD homozygote for the APOE3 Christchurch (APOECh) variant allowed us to discern common features. The male remained cognitively intact until 67 years of age despite carrying a PSEN1-E280A mutation. Like the APOECh carrier, he had extremely elevated amyloid plaque burden and limited entorhinal Tau tangle burden. He did not carry the APOECh variant but was heterozygous for a rare variant in RELN (H3447R, termed COLBOS after the Colombia-Boston biomarker research study), a ligand that like apolipoprotein E binds to the VLDLr and APOEr2 receptors. RELN-COLBOS is a gain-of-function variant showing stronger ability to activate its canonical protein target Dab1 and reduce human Tau phosphorylation in a knockin mouse. A genetic variant in a case protected from ADAD suggests a role for RELN signaling in resilience to dementia.

Expert Panel Curation of 113 Primary Mitochondrial Disease Genes for the Leigh Syndrome Spectrum.

OBJECTIVE: Primary mitochondrial diseases (PMDs) are heterogeneous disorders caused by inherited mitochondrial dysfunction. Classically defined neuropathologically as subacute necrotizing encephalomyelopathy, Leigh syndrome spectrum (LSS) is the most frequent manifestation of PMD in children, but may also present in adults. A major challenge for accurate diagnosis of LSS in the genomic medicine era is establishing gene-disease relationships (GDRs) for this syndrome with >100 monogenic causes across both nuclear and mitochondrial genomes. METHODS: The Clinical Genome Resource (ClinGen) Mitochondrial Disease Gene Curation Expert Panel (GCEP), comprising 40 international PMD experts, met monthly for 4 years to review GDRs for LSS. The GCEP standardized gene curation for LSS by refining the phenotypic definition, modifying the ClinGen Gene-Disease Clinical Validity Curation Framework to improve interpretation for LSS, and establishing a scoring rubric for LSS. RESULTS: The GDR with LSS across the nuclear and mitochondrial genomes was classified as definitive for 31 of 114 GDRs curated (27%), moderate for 38 (33%), limited for 43 (38%), and disputed for 2 (2%). Ninety genes were associated with autosomal recessive inheritance, 16 were maternally inherited, 5 were autosomal dominant, and 3 were X-linked. INTERPRETATION: GDRs for LSS were established for genes across both nuclear and mitochondrial genomes. Establishing these GDRs will allow accurate variant interpretation, expedite genetic diagnosis of LSS, and facilitate precision medicine, multisystem organ surveillance, recurrence risk counseling, reproductive choice, natural history studies, and determination of eligibility for interventional clinical trials. ANN NEUROL 2023;94:696-712.

Combined low-pass whole genome and targeted sequencing in liquid biopsies for pediatric solid tumors.

We designed a liquid biopsy (LB) platform employing low-pass whole genome sequencing (LP-WGS) and targeted sequencing of cell-free (cf) DNA from plasma to detect genome-wide copy number alterations (CNAs) and gene fusions in pediatric solid tumors. A total of 143 plasma samples were analyzed from 19 controls and 73 patients, including 44 bone or soft-tissue sarcomas and 12 renal, 10 germ cell, five hepatic, and two thyroid tumors. cfDNA was isolated from plasma collected at diagnosis, during and after therapy, and/or at relapse. Twenty-six of 37 (70%) patients enrolled at diagnosis without prior therapy (radiation, surgery, or chemotherapy) had circulating tumor DNA (ctDNA), based on the detection of CNAs from LP-WGS, including 18 of 27 (67%) patients with localized disease and eight of 10 (80%) patients with metastatic disease. None of the controls had detectable somatic CNAs. There was a high concordance of CNAs identified by LP-WGS to CNAs detected by chromosomal microarray analysis in the matching tumors. Mutations identified in tumor samples with our next-generation sequencing (NGS) panel, OncoKids®, were also detected by LP-WGS of ctDNA in 14 of 26 plasma samples. Finally, we developed a hybridization-based capture panel to target EWSR1 and FOXO1 fusions from patients with Ewing sarcoma or alveolar rhabdomyosarcoma (ARMS), respectively. Fusions were detected in the plasma from 10 of 12 patients with Ewing sarcoma and in two of two patients with ARMS. Combined, these data demonstrate the clinical applicability of our LB platform to evaluate pediatric patients with a variety of solid tumors.

Mitochondrial DNA haplogroup, genetic ancestry, and susceptibility to Ewing sarcoma.

Based on current studies, the incidence of Ewing sarcoma (ES) varies significantly by race and ethnicity, with the disease being most common in patients of European ancestry. However, race/ethnicity has generally been self-reported rather than formally evaluated at a population level using DNA evidence. Additionally, mitochondrial dysfunction is a hallmark of ES, yet there have been no reported studies of mitochondrial genetics in ES. Thus, we evaluated both the mitochondrial and nuclear ancestries of 420 pediatric ES patients in the United States using whole-genome sequencing. We found that the mitochondrial DNA (mtDNA) genomes of only six (1.4 %) patients belonged to African L haplogroups, while those of 90 % of the patients belonged to macrohaplogroup R, which includes haplogroup H, the most common maternal lineage in Europe. Compared to the general US population, European haplogroups were significantly enriched in ES patients (p < 2.2e-16) and the African haplogroups are significantly impoverished (p < 4.6e-16). Using the ancestry informative markers defined in a National Genographic study, the vast majority of patients exhibited significant nuclear ancestry originating from the Mediterranean, Northern Europe, and Southwest Asia, including all six patients with African L mtDNAs. Very few had primarily African nuclear ancestry. This is the first genomic epidemiology study to simultaneously interrogate the mitochondrial and nuclear ancestries of ES patients. While supporting previous findings of enriched European ancestry in ES patients, these results also suggest alternative hypotheses for the significant contribution of mitochondrial ancestry in ES patients, as well as the protective role of African ancestry.

Potential of Aqueous Humor as a Liquid Biopsy for Uveal Melanoma.

Tumor biopsy can identify prognostic biomarkers for metastatic uveal melanoma (UM), however aqueous humor (AH) liquid biopsy may serve as an adjunct. This study investigated whether the AH of UM eyes has sufficient circulating tumor DNA (ctDNA) to perform genetic analysis. This is a case series of 37 AH samples, taken before or after radiation, and one tumor wash sample, from 12 choroidal and 8 ciliary body (CB) melanoma eyes. AH was analyzed for nucleic acid concentrations. AH DNA and one tumor wash sample underwent shallow whole-genome sequencing followed by Illumina sequencing to detect somatic copy number alterations (SCNAs). Four post-radiation AH underwent targeted sequencing of BAP1 and GNAQ genes. Post-radiation AH had significantly higher DNA and miRNA concentrations than paired pre-radiation samples. Highly recurrent UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 post-radiation CB AH. SCNAs were highly concordant in a CB post-radiation AH with its matched tumor (r = 0.978). BAP1 or GNAQ variants were detected in 3/4 post-radiation AH samples. AH is a source of ctDNA in UM eyes, particularly in post-radiation CB eyes. For the first time, UM SCNAs and mutations were identified in AH-derived ctDNA. Suggesting that AH can serve as a liquid biopsy for UM.

Mitochondrial 1555 G>A variant as a potential risk factor for childhood glioblastoma.

Background: Childhood glioblastoma multiforme (GBM) is a highly aggressive disease with low survival, and its etiology, especially concerning germline genetic risk, is poorly understood. Mitochondria play a key role in putative tumorigenic processes relating to cellular oxidative metabolism, and mitochondrial DNA variants were not previously assessed for association with pediatric brain tumor risk. Methods: We conducted an analysis of 675 mitochondrial DNA variants in 90 childhood GBM cases and 2789 controls to identify enrichment of mitochondrial variant associated with GBM risk. We also performed this analysis for other glioma subtypes including pilocytic astrocytoma. Nuclear-encoded mitochondrial gene variants were also analyzed. Results: We identified m1555 A>G was significantly associated with GBM risk (adjusted OR 29.30, 95% CI 5.25-163.4, P-value 9.5 X 10-4). No association was detected for other subtypes. Haplotype analysis further supported the independent risk contributed by m1555 G>A, instead of a haplogroup joint effect. Nuclear-encoded mitochondrial gene variants identified significant associations in European (rs62036057 in WWOX, adjusted OR = 2.99, 95% CI 1.88-4.75, P-value = 3.42 X 10-6) and Hispanic (rs111709726 in EFHD1, adjusted OR = 3.57, 95% CI 1.99-6.40, P-value = 1.41 X 10-6) populations in ethnicity-stratified analyses. Conclusion: We report for the first time a potential role played by a functional mitochondrial ribosomal RNA variant in childhood GBM risk, and a potential role for both mitochondrial and nuclear-mitochondrial DNA polymorphisms in GBM tumorigenesis. These data implicate cellular oxidative metabolic capacity as a contributor to the etiology of pediatric glioblastoma.

m.3685T > C is a novel mitochondrial DNA variant that causes Leigh syndrome.

Variants in the mitochondrial genome can result in dysfunction of Complex I within the electron transport chain, thus causing disruptions in oxidative phosphorylation. Pathogenic variants in the MT-ND1 (NADH:ubiquinone oxidoreductase core subunit 1) gene that result in Complex I dysfunction are a known cause of Leigh syndrome. The patient is a 4-yr-old female who initially presented with generalized tonic-clonic seizures, with other symptoms of Leigh syndrome becoming apparent after the seizures. A three-generation pedigree revealed no family history of mitochondrial disorders. Laboratory studies were remarkable for elevated blood lactate, alanine, and GDF15. T2-weighted magnetic resonance imaging (MRI) revealed bilateral asymmetric signal hyperintensities in the basal ganglia, specifically in the bilateral putamen and right caudate. Magnetic resonance spectroscopy showed regionally elevated glucose and lactate. Mitochondrial respiratory chain enzyme analysis on skin fibroblasts demonstrated slightly reduced Complex I function. A 16-gene dystonia panel and chromosomal microarray analysis did not identify any disease-causing variants. Combined exome and mitochondrial genome sequencing identified the m.3685T > C (MT-ND1 p.Tyr127His) variant with 62.3% heteroplasmy with no alternative cause for the patient's condition. Mitochondrial genome sequencing of the mother demonstrated that the m.3685T > C variant occurred de novo. The m.3685T > C variant is absent from population databases. The tyrosine 127 residue is highly conserved, and several nearby pathogenic variants in the MT-ND1 gene have been previously associated with Leigh syndrome. We propose that the m.3685T > C variant is a novel mitochondrial DNA variant that causes Leigh syndrome, and we classify this variant as likely pathogenic based on currently available information.

Aqueous Humor as a Liquid Biopsy for Retinoblastoma: Clear Corneal Paracentesis and Genomic Analysis.

There is significant potential clinical utility for the application of a liquid biopsy platform for retinoblastoma, given that direct tumor biopsy is prohibited in these patients. The aqueous humor (AH) forms in a separate compartment from the tumor but is enclosed within the same ocular space. Thus, it is an enriched source of eye-specific tumoral genomic information that can be used as a liquid biopsy or surrogate to tumor biopsy for this disease. This manuscript details a methodology for safely extracting the AH from retinoblastoma eyes via clear corneal paracentesis. Additionally, the steps for genomic analysis, including cell-free DNA isolation and purification, next-generation sequencing, somatic copy number alteration (SCNA) analysis, RB1 single nucleotide variant (SNV) mutation identification, and tumor fraction estimation are presented. The pre-analytical, analytical, and early clinical validity of the AH liquid biopsy platform have been evaluated; however, it is not without limitations. These are largely a consequence of the quantity of cell-free DNA that is required for certain steps of the assay. Compared to other blood-based liquid biopsy platforms currently under investigation for retinoblastoma, an AH-based platform is limited by the volume of biofluid (and thus the quantity of DNA) that can be extracted from the eye; the benefit is that AH is eye-specific. The platform discussed here is unique in that it detects circulating tumor DNA in the AH via two mechanisms (SCNAs and RB1 SNVs), yielding a higher sensitivity for identifying tumoral genomic information. The AH liquid biopsy has the potential for direct clinical application to precision oncology for retinoblastoma patients, with particular importance for patients with bilateral disease as the AH is specific to the tumors in each eye. There is ongoing research with applications of this platform to patients with other ocular tumors as well.

The spectrum of mitochondrial DNA (mtDNA) mutations in pediatric CNS tumors.

BACKGROUND: We previously established the landscape of mitochondrial DNA (mtDNA) mutations in 23 subtypes of pediatric malignancies, characterized mtDNA mutation profiles among these subtypes, and provided statistically significant evidence for a contributory role of mtDNA mutations to pediatric malignancies. METHODS: To further delineate the spectrum of mtDNA mutations in pediatric central nervous system (CNS) tumors, we analyzed 545 tumor-normal paired whole-genome sequencing datasets from the Children's Brain Tumor Tissue Consortium. RESULTS: Germline mtDNA variants were used to determine the haplogroup, and maternal ancestry, which was not significantly different among tumor types. Among 166 (30.5%) tumors we detected 220 somatic mtDNA mutations, primarily missense mutations (36.8%), as well as 22 loss-of-function mutations. Different pediatric CNS tumor subtypes had distinct mtDNA mutation profiles. The number of mtDNA mutations per tumor ranged from 0.20 (dysembryoplastic neuroepithelial tumor [DNET]) to 0.75 (meningiomas). The average heteroplasmy was 10.7%, ranging from 4.6% in atypical teratoid/rhabdoid tumor (AT/RT) to 26% in diffuse intrinsic pontine glioma. High-grade gliomas had a significant higher number of mtDNA mutations per sample than low-grade gliomas (0.6 vs 0.27) (P = .004), with almost twice as many missense mtDNA mutations per sample (0.24 vs 0.11), and higher average heteroplasmy levels (16% vs 10%). Recurrent mtDNA mutations may represent hotspots which may serve as biologic markers of disease. CONCLUSIONS: Our findings demonstrate varying contributions of mtDNA mutations in different subtypes of CNS tumors. Sequencing the mtDNA genome may ultimately be used to characterize CNS tumors at diagnosis and monitor disease progression.

Inter-eye genomic heterogeneity in bilateral retinoblastoma via aqueous humor liquid biopsy.

Germline alterations in the RB1 tumor suppressor gene predispose patients to develop retinoblastoma (RB) in both eyes. While similar treatment is given for each eye, there is often a variable therapeutic response between the eyes. Herein, we use the aqueous humor (AH) liquid biopsy to evaluate the cell-free tumor DNA (ctDNA) from each eye in a patient with bilateral RB. Despite the same predisposing germline RB1 mutation, AH analysis identified a different somatic RB1 mutation as well as separate and distinct chromosomal alterations in each eye. The longitudinal alterations in tumor fraction (TFx) corresponded to therapeutic responses in each eye. This case demonstrates that bilateral RB tumors develop separate genomic alterations, which may play a role in tumorigenesis and prognosis for eye salvage. Identifying these inter-eye differences without the need for enucleated tumor tissue may help direct active management of RB, with particular usefulness in bilateral cases.