Genomic profiling of pediatric hematologic malignancies and diagnosis of cancer predisposition syndromes: tumor-only versus paired tumor-normal sequencing.

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Uncovering the Genetic Etiology of Inherited Bone Marrow Failure Syndromes Using a Custom-Designed Next-Generation Sequencing Panel.

Inherited bone marrow failure syndromes (IBMFS) are a group of heterogeneous disorders that account for ∼30% of pediatric cases of bone marrow failure and are often associated with developmental abnormalities and cancer predisposition. This article reports the laboratory validation and clinical utility of a large-scale, custom-designed next-generation sequencing panel, Children's Hospital of Philadelphia (CHOP) IBMFS panel, for the diagnosis of IBMFS in a cohort of pediatric patients. This panel demonstrated excellent analytic accuracy, with 100% sensitivity, ≥99.99% specificity, and 100% reproducibility on validation samples. In 269 patients with suspected IBMFS, this next-generation sequencing panel was used for identifying single-nucleotide variants, small insertions/deletions, and copy number variations in mosaic or nonmosaic status. Sixty-one pathogenic/likely pathogenic variants (54 single-nucleotide variants/insertions/deletions and 7 copy number variations) and 24 hypomorphic variants were identified, resulting in the molecular diagnosis of IBMFS in 21 cases (7.8%) and exclusion of IBMFS with a diagnosis of a blood disorder in 10 cases (3.7%). Secondary findings, including evidence of early hematologic malignancies and other hereditary cancer-predisposition syndromes, were observed in 9 cases (3.3%). The CHOP IBMFS panel was highly sensitive and specific, with a significant increase in the diagnostic yield of IBMFS. These findings suggest that next-generation sequencing-based panel testing should be a part of routine diagnostics in patients with suspected IBMFS.

Novel ATXN1/ATXN1L::NUTM2A fusions identified in aggressive infant sarcomas with gene expression and methylation patterns similar to CIC-rearranged sarcoma.

CIC-rearranged sarcomas are newly defined undifferentiated soft tissue tumors with CIC-associated fusions, and dismal prognosis. CIC fusions activate PEA3 family genes, ETV1/4/5, leading to tumorigenesis and progression. We report two high-grade CNS sarcomas of unclear histological diagnosis and one disseminated tumor of unknown origin with novel fusions and similar gene-expression/methylation patterns without CIC rearrangement. All three patients were infants with aggressive diseases, and two experienced rapid disease deterioration and death. Whole-transcriptome sequencing identified an ATXN1-NUTM2A fusion in the two CNS tumors and an ATXN1L-NUTM2A fusion in case 3. ETV1/4/5 and WT1 overexpression were observed in all three cases. Methylation analyses predicted CIC-rearranged sarcoma for all cases. Retrospective IHC staining on case 2 demonstrated ETV4 and WT1 overexpression. ATXN1 and ATXN1L interact with CIC forming a transcription repressor complex. We propose that ATXN1/ATXN1L-associated fusions disrupt their interaction with CIC and decrease the transcription repressor complex, leading to downstream PEA3 family gene overexpression. These three cases with novel ATXN1/ATXN1L-associated fusions and features of CIC-rearranged sarcomas may further expand the scope of "CIC-rearranged" sarcomas to include non-CIC rearrangements. Additional cases are needed to demonstrate if ATXN1/ATXN1L-NUTM2A fusions are associated with younger age and more aggressive diseases.

A novel TP53 tandem duplication in a child with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is one of the most common cancer predisposition syndromes that affects both children and adults. Individuals with LFS are at an increased risk of developing various types of cancer over their lifetime including soft tissue sarcomas, osteosarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Heterozygous germline pathogenic variants in the tumor suppressor gene TP53 are the known causal genetic defect for LFS. Single-nucleotide variants (SNVs) including missense substitutions that occur in the highly conserved DNA binding domain of the protein are the most common alterations, followed by nonsense and splice site variants. Gross copy-number changes in TP53 are rare and account for <1% of all variants. Using next-generation sequencing (NGS) panels, we identified a paternally inherited germline intragenic duplication of TP53 in a child with metastatic osteosarcoma who later developed acute myeloid leukemia (AML). Transcriptome sequencing (RNA-seq) demonstrated the duplication was tandem, encompassing exons 2-6 and 28 nt of the untranslated region (UTR) upstream of the start codon in exon 2. The inclusion of the 28 nt is expected to result in a frameshift with a stop codon 18 codons downstream from the exon 6, leading to a loss-of-function allele. This case highlights the significance of simultaneous identification of both significant copy-number variants as well as SNVs/indels using NGS panels.

Atypical teratoid rhabdoid tumor in a child with neurofibromatosis type 2: A novel dual diagnosis.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the development of tumors of the nervous system and is associated with NF2 gene alterations. Atypical teratoid rhabdoid tumor (ATRT) is a malignant central nervous system tumor that occurs primarily in children less than 3 years of age. The majority of cases of ATRT demonstrate genomic alterations of SMARCB1, a core member of the SWI/SNF chromatin-remodeling complex and tumor suppressor gene. SMARCB1 inactivation in ATRT is occasionally associated with somatic NF2 deletion; however, concurrent germline NF2 mutations have not been reported. Herein, we describe the case of a 3-year-old patient who presented with an intracranial mass. Next generation sequencing analysis of tumor identified homozygous deletions of the entire SMARCB1 gene and exon 7 to exon 14 of NF2 gene with whole chromosome 22 loss of heterozygosity (LOH). Multiplex Ligation-dependent Probe Amplification (MLPA) assay performed on blood identified a germline heterozygous intragenic deletion of NF2 exon 7 to exon 14; a somatic chromosome 22 LOH led to the homozygous deletion. SMARCB1 MLPA assay of blood showed no deletion. This cascade represents a novel, "four-hit" mechanism of SMARCB1 inactivation resulting in ATRT and the first known dual diagnosis of NF2 and ATRT.

Advanced approach for comprehensive mtDNA genome testing in mitochondrial disease.

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test ('MitoGenome') using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations.

Genomic characterization of a PPP1CB-ALK fusion with fusion gene amplification in a congenital glioblastoma.

ALK (Anaplastic lymphoma kinase) fusion proteins are oncogenic and have been seen in various tumors. PPP1CB-ALK fusions are rare but have been reported in a few patients with low- or high-grade gliomas. However, little is known regarding the mechanism of fusion formation and genomic break points of this fusion. We performed genomic characterization of a PPP1CB-ALK fusion with fusion gene amplification in a congenital glioblastoma. The PPP1CB-ALK consists of exons 1-5 of PPP1CB and exons 20-29 of ALK. The genomic translocation breakpoints were determined by real-time quantitative PCR (RT-qPCR) and Sanger sequencing of genomic DNA. Next generation sequencing, RT-qPCR and fluorescence in situ hybridization analyses demonstrated PPP1CB-ALK amplification. Copy number analyses of genes between PPP1CB and ALK using RT-qPCR suggest that the PPP1CB-ALK is likely the result of local chromothripsis followed by episomal amplification. Transcriptome sequencing demonstrated high-level SOX2 expression and predicted WNT/ß-catenin pathway activation, suggesting possible therapeutic approaches.

AUDIOME: a tiered exome sequencing-based comprehensive gene panel for the diagnosis of heterogeneous nonsyndromic sensorineural hearing loss.

PURPOSE: Hereditary hearing loss is highly heterogeneous. To keep up with rapidly emerging disease-causing genes, we developed the AUDIOME test for nonsyndromic hearing loss (NSHL) using an exome sequencing (ES) platform and targeted analysis for the curated genes. METHODS: A tiered strategy was implemented for this test. Tier 1 includes combined Sanger and targeted deletion analyses of the two most common NSHL genes and two mitochondrial genes. Nondiagnostic tier 1 cases are subjected to ES and array followed by targeted analysis of the remaining AUDIOME genes. RESULTS: ES resulted in good coverage of the selected genes with 98.24% of targeted bases at >15 ×. A fill-in strategy was developed for the poorly covered regions, which generally fell within GC-rich or highly homologous regions. Prospective testing of 33 patients with NSHL revealed a diagnosis in 11 (33%) and a possible diagnosis in 8 cases (24.2%). Among those, 10 individuals had variants in tier 1 genes. The ES data in the remaining nondiagnostic cases are readily available for further analysis. CONCLUSION: The tiered and ES-based test provides an efficient and cost-effective diagnostic strategy for NSHL, with the potential to reflex to full exome to identify causal changes outside of the AUDIOME test.

Fabrication and properties of thermosensitive organic/inorganic hybrid hydrogel thin films.

We report on a facile method for fabricating thermosensitive organic/inorganic hybrid hydrogel thin films from a cross-linkable organic/inorganic hydrid copolymer, poly[ N-isopropylacrylamide- co-3-(trimethoxysilyl)propylmethacrylate] [P(NIPAm- co-TMSPMA)]. Fourier transform infrared (FT-IR) spectra confirmed the formation of hybrid hydrogel thin films after hydrolysis of the methoxysilyl groups (Si-O-CH 3) and subsequent condensation of the silanol groups (Si-OH). Atomic force microscopy (AFM) images revealed that the surface morphology of the hydrogel thin films depended on the supporting substrates. Microdomains were observed for the hydrogel thin films on a gold surface, which can be attributed to inhomogeneous network structures. The thermoresponsive swelling-deswelling behavior and the viscoelastic properties of the hydrogel thin films were investigated as a function of temperature (25-45 degrees C) by using a quartz crystal microbalance (QCM) operated in water. The high frequency shear modulus of the P(NIPAm- co-TMPSMA) hydrogel thin films was several hundred kilopascals.