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.

High Prevalence of SARS-CoV-2 Genetic Variation and D614G Mutation in Pediatric Patients With COVID-19.

BACKGROUND: The full spectrum of the disease phenotype and viral genotype of coronavirus disease 2019 (COVID-19) have yet to be thoroughly explored in children. Here, we analyze the relationships between viral genetic variants and clinical characteristics in children. METHODS: Whole-genome sequencing was performed on respiratory specimens collected for all SARS-CoV-2-positive children (n = 141) between March 13 and June 16, 2020. Viral genetic variations across the SARS-CoV-2 genome were identified and investigated to evaluate genomic correlates of disease severity. RESULTS: Higher viral load was detected in symptomatic patients (P = .0007) and in children <5 years old (P = .0004). Genomic analysis revealed a mean pairwise difference of 10.8 single nucleotide variants (SNVs), and the majority (55.4%) of SNVs led to an amino acid change in the viral proteins. The D614G mutation in the spike protein was present in 99.3% of the isolates. The calculated viral mutational rate of 22.2 substitutions/year contrasts the 13.5 substitutions/year observed in California isolates without the D614G mutation. Phylogenetic clade 20C was associated with severe cases of COVID-19 (odds ratio, 6.95; P = .0467). Epidemiological investigation revealed major representation of 3 of 5 major Nextstrain clades (20A, 20B, and 20C) consistent with multiple introductions of SARS-CoV-2 in Southern California. CONCLUSIONS: Genomic evaluation demonstrated greater than expected genetic diversity, presence of the D614G mutation, increased mutation rate, and evidence of multiple introductions of SARS-CoV-2 into Southern California. Our findings suggest a possible association of phylogenetic clade 20C with severe disease, but small sample size precludes a definitive conclusion. Our study warrants larger and multi-institutional genomic evaluation and has implications for infection control practices.

Increased viral variants in children and young adults with impaired humoral immunity and persistent SARS-CoV-2 infection: A consecutive case series.

BACKGROUND: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. METHODS: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. FINDINGS: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. INTERPRETATION: Our results highlight the potential need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.

Persistent SARS-CoV-2 infection and increasing viral variants in children and young adults with impaired humoral immunity.

Background: There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. Methods: We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. Findings: We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. Interpretation: Our results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered. Funding: The work was partially funded by The Saban Research Institute at Children's Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

Early pandemic molecular diversity of SARS-CoV-2 in children.

Background: In the US, community circulation of the SARS-CoV-2 virus likely began in February 2020 after mostly travel-related cases. Children's Hospital of Philadelphia began testing on 3/9/2020 for pediatric and adult patients, and for all admitted patients on 4/1/2020, allowing an early glimpse into the local molecular epidemiology of the virus. Methods: We obtained 169 SARS-CoV-2 samples (83 from patients <21 years old) from March through May and produced whole genome sequences. We used genotyping tools to track variants over time and to test for possible genotype associated clinical presentations and outcomes in children. Results: Our analysis uncovered 13 major lineages that changed in relative abundance as cases peaked in mid-April in Philadelphia. We detected at least 6 introductions of distinct viral variants into the population. As a group, children had more diverse virus genotypes than the adults tested. No strong differences in clinical variables were associated with genotypes. Conclusions: Whole genome analysis revealed unexpected diversity, and distinct circulating viral variants within the initial peak of cases in Philadelphia. Most introductions appeared to be local from nearby states. Although limited by sample size, we found no evidence that different genotypes had different clinical impacts in children in this study.

Detection of mitochondrial DNA variants at low level heteroplasmy in pediatric CNS and extra-CNS solid tumors with three different enrichment methods.

The mitochondrial genome is small, 16.5 kb, and yet complex to study due to an abundance of mitochondria in any given cell or tissue. Mitochondrial DNA (mtDNA) mutations have been previously described in cancer, many of which were detected at low heteroplasmy. In this study we enriched the mitochondrial genome in primary pediatric tumors for detection of mtDNA variants. We completed mtDNA enrichment using REPLI-g, Agilent SureSelect, and long-range polymerase chain reaction (LRPCR) followed by next generation sequencing (NGS) on Illumina platforms. Primary tumor and germline genomic DNA from a variety of pediatric central nervous system (CNS) and extra-CNS solid tumors were analyzed by the three different methods. Although all three methods performed equally well for detecting variants at high heteroplasmy or homoplasmy, only LRPCR and SureSelect-based enrichment methods provided consistent results for variants that were present at less than five percent heteroplasmy. We then applied both LRPCR and SureSelect to three successive samples from a patient with multiply-recurrent gliofibroma and detected a low-level novel mutation as well as a change in heteroplasmy levels of a synonymous variant that was correlated with progression of disease. IMPLICATION: This study demonstrates that LRPCR and SureSelect enrichment, but not REPLI-g, followed by NGS are accurate methods for studying the mtDNA variations at low heteroplasmy, which may be applied to studying mtDNA mutations in cancer.

OncoKids: A Comprehensive Next-Generation Sequencing Panel for Pediatric Malignancies.

The OncoKids panel is an amplification-based next-generation sequencing assay designed to detect diagnostic, prognostic, and therapeutic markers across the spectrum of pediatric malignancies, including leukemias, sarcomas, brain tumors, and embryonal tumors. This panel uses low input amounts of DNA (20 ng) and RNA (20 ng) and is compatible with formalin-fixed, paraffin-embedded and frozen tissue, bone marrow, and peripheral blood. The DNA content of this panel covers the full coding regions of 44 cancer predisposition loci, tumor suppressor genes, and oncogenes; hotspots for mutations in 82 genes; and amplification events in 24 genes. The RNA content includes 1421 targeted gene fusions. We describe the validation of this panel by using a large cohort of 192 unique clinical samples that included a wide range of tumor types and alterations. Robust performance was observed for analytical sensitivity, reproducibility, and limit of detection studies. The results from this study support the use of OncoKids for routine clinical testing of a wide variety of pediatric malignancies.

Clinical utility of the low-density Infinium QC genotyping Array in a genomics-based diagnostics laboratory.

BACKGROUND: With 15,949 markers, the low-density Infinium QC Array-24 BeadChip enables linkage analysis, HLA haplotyping, fingerprinting, ethnicity determination, mitochondrial genome variations, blood groups and pharmacogenomics. It represents an attractive independent QC option for NGS-based diagnostic laboratories, and provides cost-efficient means for determining gender, ethnic ancestry, and sample kinships, that are important for data interpretation of NGS-based genetic tests. METHODS: We evaluated accuracy and reproducibility of Infinium QC genotyping calls by comparing them with genotyping data of the same samples from other genotyping platforms, whole genome/exome sequencing. Accuracy and robustness of determining gender, provenance, and kinships were assessed. RESULTS: Concordance of genotype calls between Infinium QC and other platforms was above 99%. Here we show that the chip's ancestry informative markers are sufficient for ethnicity determination at continental and sometimes subcontinental levels, with assignment accuracy varying with the coverage for a particular region and ethnic groups. Mean accuracies of provenance prediction at a regional level were varied from 81% for Asia, to 89% for Americas, 86% for Africa, 97% for Oceania, 98% for Europe, and 100% for India. Mean accuracy of ethnicity assignment predictions was 63%. Pairwise concordances of AFR samples with the samples from any other super populations were the lowest (0.39-0.43), while the concordances within the same population were relatively high (0.55-0.61). For all populations except African, cross-population comparisons were similar in their concordance ranges to the range of within-population concordances (0.54-0.57). Gender determination was correct in all tested cases. CONCLUSIONS: Our results indicate that the Infinium QC Array-24 chip is suitable for cost-efficient, independent QC assaying in the settings of an NGS-based molecular diagnostic laboratory; hence, we recommend its integration into the standard laboratory workflow. Low-density chips can provide sample-specific measures for variant call accuracy, prevent sample mix-ups, validate self-reported ethnicities, and detect consanguineous cases. Integration of low-density chips into QC procedures aids proper interpretation of candidate sequence variants. To enhance utility of this low-density chip, we recommend expansion of ADME and mitochondrial markers. Inexpensive Infinium-like low-density human chips have a potential to become a "Swiss army knife" among genotyping assays suitable for many applications requiring high-throughput assays.