Molecular Diagnosis of Mosaic Overgrowth Syndromes Using a Custom-Designed Next-Generation Sequencing Panel.

Recent studies have discovered a group of overgrowth syndromes, such as congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) syndrome, Proteus syndrome, and megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, are caused by somatic activating variants in genes involved in the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway. Because of the low-abundance nature of these variants, Sanger sequencing often yields negative results. We have developed and validated a next-generation sequencing (NGS) panel that targets all known variants associated with these syndromes. Fifty cases, including two prenatal cases, were tested using the panel. A pathogenic variant in the PIK3CA, PIK3R2, or AKT1 gene was identified in 28 of the 50 cases with the variant allele frequencies ranging from 1.0% to 49.2%. These variants were only present in the affected tissues in most of the cases, demonstrating a causal role in the development of these diseases. In vitro cell culture showed significant enrichment of the cells harboring variant alleles, suggesting that these variants render growth advantages to mutant cells. Phenotype-genotype correlation analysis showed PIK3CA mutation hotspots at residues E542, E545, and H1047 are often associated with CLOVES syndrome, whereas PIK3CA G914R is preferentially related to MCAP. We thus demonstrate that NGS technology is highly sensitive for detecting low-level mosaicism and can facilitate clinical diagnosis of mosaic overgrowth syndromes in both prenatal and postnatal settings.

Integrating Molecular Testing in the Diagnosis and Management of Children with Thyroid Lesions.

Thyroid nodules occur in 1-2% of children, and identifying which nodules are malignant is often challenging. Cytologic evaluation facilitates the diagnosis of thyroid lesions (TLs), but in 10-40% of cases the interpretation is indeterminate. Patients with indeterminate diagnoses are often treated with hemithyroidectomy followed by completion thyroidectomy, if cancer is found in the initial specimen. Exposing patients to multiple surgeries increases costs and morbidity. The American Thyroid Association states that a combination of molecular markers is likely to optimize the management of patients with indeterminate cytology. However, few studies have addressed the molecular alterations present in pediatric TL. Twenty-seven thyroid carcinomas from patients 10 to 19 years of age were tested for alterations common in adult TL, including BRAF V600E mutation, RET fusions, and TERT promoter mutations. Mutation-negative cases were subsequently analyzed with a next-generation sequencing (NGS) mutation panel to search for additional targets. Histologic diagnoses included 12 classic papillary thyroid carcinomas (PTCs), 13 follicular variant PTCs, 1 medullary thyroid carcinoma, and 1 follicular carcinoma. Fourteen cases showed lymph node involvement, and 13 cases demonstrated lymphovascular invasion. The BRAF V600E mutation was detected in 10/27 cases, and RET fusions were detected in 6/27 cases. No TERT promoter mutations were identified in any of the cases. The NGS panel revealed additional RET and CTNNB1 pathogenic missense mutations. Our results demonstrate that molecular abnormalities are common in pediatric TLs and suggest that incorporation of molecular testing will be helpful in optimizing patient management.

Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney.

The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

Copy number variation in the horse genome.

We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches.

Lipid oxidation and modification of amyloid-ß (Aß) in vitro and in vivo.

Oxidative damage and amyloid-ß (Aß) protein misfolding are prominent features of Alzheimer's disease (AD). In vitro studies indicated a direct linkage between these two features, where lipid oxidation products augmented Aß misfolding. We tested this linkage further, mimicking specific conditions present in amyloid plaques. In vitro lipid oxidation and lipid modification of Aß were thus performed with elevated levels of copper or physiological levels of calcium. These in vitro experiments were then confirmed by in vivo immunohistochemical and chemical tagging of oxidative damage in brains from the PSAPP mouse model of AD. Our in vitro findings indicate that: 1) high levels of copper prevent lipid oxidation; 2) physiological concentrations of calcium reduce 4 hydroxy-2-nonenal (HNE) modification of Aß; and 3) anti-Aß and HNE antibody epitopes are differentially masked. In vivo we demonstrated increased lipid oxidation around plaques but 4) a lack of immunological colocalization of HNE-adducts with Aß. Thus, the lack of colocalization of Aß and HNE-adduct immunostaining is most likely due to a combination of metals inhibiting HNE modification of Aß, quenching lipid oxidation and a masking of HNE-Aß histopathology. However, other forms of oxidative damage colocalize with Aß in plaques, as demonstrated using a chemical method for identifying oxidative damage. Additionally, these findings suggest that HNE modification of Aß may affect therapeutic antibodies targeting the amino terminal of Aß and that metals effect on lipid oxidation and lipid modification of Aß could raise concerns on emerging anti-AD treatments with metal chelators.

A new class of silica cross-linked micellar core-shell nanoparticles.

Micellar nanoparticles made of surfactants and polymers have attracted wide attention in the materials and biomedical community for controlled drug delivery, molecular imaging, and sensing; however, their long-term stability remains a topic of intense study. Here we report a new class of robust, ultrafine silica core-shell nanoparticles formed from silica cross-linked, individual block copolymer micelles. Compared with pure polymeric micelles, the main advantage of the new core-shell nanoparticles is that they have significantly improved stability and do not break down during dilution. We also studied the drug loading and release properties of the silica cross-linked micellar particles, and we found that the new core-shell nanoparticles have a slower release rate which allows the entrapped molecules to be slowly released over a much longer period of time under the same experimental conditions. A range of functional groups can be easily incorporated through co-condensation with the silica matrix. The potential to deliver hydrophobic agents into cancer cells has been demonstrated. Because of their unique structures and properties, these novel core-shell nanoparticles could potentially provide a new nanomedicine platform for imaging, detection, and treatment, as well as novel colloidal particles and building blocks for mutlifunctional materials.