Comprehensive Genomic Analysis of Cemento-Ossifying Fibroma.

Cemento-ossifying fibroma (COF) of the jaws is currently classified as a benign mesenchymal odontogenic tumor, and only targeted approaches have been used to assess its genetic alterations. A minimal proportion of COFs harbor CDC73 somatic mutations, and copy number alterations (CNAs) involving chromosomes 7 and 12 have recently been reported in a small proportion of cases. However, the genetic background of COFs remains obscure. We used a combination of whole-exome sequencing and RNA sequencing to assess somatic mutations, fusion transcripts, and CNAs in a cohort of 12 freshly collected COFs. No recurrent fusions have been identified among the 5 cases successfully analyzed by RNA sequencing, with in-frame fusions being detected in 2 cases (MARS1::GOLT1B and PARG::BMS1 in one case and NCLN::FZR1 and NFIC::SAMD1 in the other case) and no candidate fusions identified for the remaining 3 cases. No recurrent pathogenic mutations were detected in the 11 cases that had undergone whole-exome sequencing. A KRAS p.L19F missense variant was detected in one case, and 2 CDC73 deletions were detected in another case. The other variants were of uncertain significance and included variants in PC, ACTB, DOK6, HACE1, and COL1A2 and previously unreported variants in PTPN14, ATP5F1C, APOBEC1, HDAC5, ATF7IP, PARP2, and ACTR3B. The affected genes do not clearly converge on any signaling pathway. CNAs were detected in 5/11 cases (45%), with copy gains involving chromosome 12 occurring in 3/11 cases (27%). In conclusion, no recurrent fusions or pathogenic variants have been detected in the present COF cohort, with copy gains involving chromosome 12 occurring in 27% of cases.

Association of novel mutation in TRPV4 with familial nonsyndromic craniosynostosis with complete penetrance and variable expressivity.

OBJECTIVE: The aim of this study was to characterize a novel pathogenic variant in the transient receptor potential vanilloid 4 (TRPV4) gene, causing familial nonsyndromic craniosynostosis (CS) with complete penetrance and variable expressivity. METHODS: Whole-exome sequencing was performed on germline DNA of a family with nonsyndromic CS to a mean depth coverage of 300× per sample, with greater than 98% of the targeted region covered at least 25×. In this study, the authors detected a novel variant, c.496C>A in TRPV4, exclusively in the four affected family members. The variant was modeled using the structure of the TRPV4 protein from Xenopus tropicalis. In vitro assays in HEK293 cells overexpressing wild-type TRPV4 or TRPV4 p.Leu166Met were used to assess the effect of the mutation on channel activity and downstream MAPK signaling. RESULTS: The authors identified a novel, highly penetrant heterozygous variant in TRPV4 (NM_021625.4:c.496C>A) causing nonsyndromic CS in a mother and all three of her children. This variant results in an amino acid change (p.Leu166Met) in the intracellular ankyrin repeat domain distant from the Ca2+-dependent membrane channel domain. In contrast to other TRPV4 mutations in channelopathies, this variant does not interfere with channel activity as identified by in silico modeling and in vitro overexpression assays in HEK293 cells. CONCLUSIONS: Based on these findings, the authors hypothesized that this novel variant causes CS by modulating the binding of allosteric regulatory factors to TRPV4 rather than directly modifying its channel activity. Overall, this study expands the genetic and functional spectrum of TRPV4 channelopathies and is particularly relevant for the genetic counseling of CS patients.

Drosophila Tet Is Required for Maintaining Glial Homeostasis in Developing and Adult Fly Brains.

Ten-eleven translocation (TET) proteins are crucial epigenetic regulators highly conserved in multicellular organisms. TETs' enzymatic function in demethylating 5-methyl cytosine in DNA is required for proper development and TETs are frequently mutated in cancer. Recently, Drosophila melanogaster Tet (dTet) was shown to be highly expressed in developing fly brains and discovered to play an important role in brain and muscle development as well as fly behavior. Furthermore, dTet was shown to have different substrate specificity compared with mammals. However, the exact role dTet plays in glial cells and how ectopic TET expression in glial cells contributes to tumorigenesis and glioma is still not clear. Here, we report a novel role for dTet specifically in glial cell organization and number. We show that loss of dTet affects the organization of a specific glia population in the optic lobe, the "optic chiasm" glia. Additionally, we find irregularities in axon patterns in the ventral nerve cord (VNC) both, in the midline and longitudinal axons. These morphologic glia and axonal defects were accompanied by locomotor defects in developing larvae escalating to immobility in adult flies. Furthermore, glia homeostasis was disturbed in dTet-deficient brains manifesting in gain of glial cell numbers and increased proliferation. Finally, we establish a Drosophila model to understand the impact of human TET3 in glia and find that ectopic expression of hTET3 in dTet-expressing cells causes glia expansion in larval brains and affects sleep/rest behavior and the circadian clock in adult flies.

De novo TRPV4 Leu619Pro variant causes a new channelopathy characterised by giant cell lesions of the jaws and skull, skeletal abnormalities and polyneuropathy.

BACKGROUND: Pathogenic germline variants in Transient Receptor Potential Vanilloid 4 Cation Channel (TRPV4) lead to channelopathies, which are phenotypically diverse and heterogeneous disorders grossly divided in neuromuscular disorders and skeletal dysplasia. We recently reported in sporadic giant cell lesions of the jaws (GCLJs) novel, somatic, heterozygous, gain-of-function mutations in TRPV4, at Met713. METHODS: Here we report two unrelated women with a de novo germline p.Leu619Pro TRPV4 variant and an overlapping systemic disorder affecting all organs individually described in TRPV4 channelopathies. RESULTS: From an early age, both patients had several lesions of the nervous system including progressive polyneuropathy, and multiple aggressive giant cell-rich lesions of the jaws and craniofacial/skull bones, and other skeletal lesions. One patient had a relatively milder disease phenotype possibly due to postzygotic somatic mosaicism. Indeed, the TRPV4 p.Leu619Pro variant was present at a lower frequency (variant allele frequency (VAF)=21.6%) than expected for a heterozygous variant as seen in the other proband, and showed variable regional frequency in the GCLJ (VAF ranging from 42% to 10%). In silico structural analysis suggests that the gain-of-function p.Leu619Pro alters the ion channel activity leading to constitutive ion leakage. CONCLUSION: Our findings define a novel polysystemic syndrome due to germline TRPV4 p.Leu619Pro and further extend the spectrum of TRPV4 channelopathies. They further highlight the convergence of TRPV4 mutations on different organ systems leading to complex phenotypes which are further mitigated by possible post-zygotic mosaicism. Treatment of this disorder is challenging, and surgical intervention of the GCLJ worsens the lesions, suggesting the future use of MEK inhibitors and TRPV4 antagonists as therapeutic modalities for unmet clinical needs.

Inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1/INI1 protein in a molecular subset of atypical teratoid/rhabdoid tumors.

Loss of nuclear SMARCB1 (INI1/hSNF5/BAF47) protein expression due to biallelic mutations of the SMARCB1 tumor suppressor gene is a hallmark of atypical teratoid/rhabdoid tumors (ATRT), but the presence of cytoplasmic SMARCB1 protein in these tumors has not yet been described. In a series of 102 primary ATRT, distinct cytoplasmic SMARCB1 staining on immunohistochemistry was encountered in 19 cases (19%) and was highly over-represented in cases showing pathogenic sequence variants leading to truncation or mutation of the C-terminal part of SMARCB1 (15/19 vs. 4/83; Chi-square: 56.04, p = 1.0E-10) and, related to this, in tumors of the molecular subgroup ATRT-TYR (16/36 vs. 3/66; Chi-square: 24.47, p = 7.6E-7). Previous reports have indicated that while SMARCB1 lacks a bona fide nuclear localization signal, it harbors a masked nuclear export signal (NES) and that truncation of the C-terminal region results in unmasking of this NES leading to cytoplasmic localization. To determine if cytoplasmic localization found in ATRT is due to unmasking of NES, we generated GFP fusions of one of the SMARCB1 truncating mutations (p.Q318X) found in the tumors along with a p.L266A mutation, which was shown to disrupt the interaction of SMARCB1-NES with exportin-1. We found that while the GFP-SMARCB1(Q318X) mutant localized to the cytoplasm, the double mutant GFP-SMARCB1(Q318X;L266A) localized to the nucleus, confirming NES requirement for cytoplasmic localization. Furthermore, cytoplasmic SMARCB1(Q318X) was unable to cause senescence as determined by morphological observations and by senescence-associated ß-galactosidase assay, while nuclear SMARCB1(Q318X;L266A) mutant regained this function. Selinexor, a selective exportin-1 inhibitor, was effective in inhibiting the nuclear export of SMARCB1(Q318X) and caused rapid cell death in rhabdoid tumor cells. In conclusion, inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1. Therapies aimed at preventing nuclear export of mutant SMARCB1 protein may represent a promising targeted therapy in ATRT harboring truncating C-terminal SMARCB1 mutations.

Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles.

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type-based diagnosis and antithrombotic intervention.

Whole-exome sequencing reveals novel vacuolar ATPase genes' variants and variants in genes involved in lysosomal biology and autophagosomal formation in oral granular cell tumors.

BACKGROUND: Granular cell tumors (GCTs) are rare neuroectodermal soft tissue neoplasms that mainly affect the skin of the upper limbs and trunks and the oral cavity. GCTs are derived from Schwann cells and, ultrastructurally, their intracytoplasmic granules are considered autophagosomes or autophagolysosomes and are consistent with myelin accumulation. METHODS: In this study, a convenience set of 22 formalin-fixed, paraffin-embedded samples of oral GCTs, all but one sample located at the tongue, was screened for mutations by whole-exome (WES) or targeted sequencing. RESULTS: WES revealed two novel variants in genes of the vacuolar ATPase (V-ATPase) complex: ATP6AP1 frameshift c.746_749del, leading to p.P249Hfs*4, and ATP6V1A non-synonymous c.G868A, leading to p.D290N. Each of these mutations occurred in one case. With regard to the samples that were wild type for these V-ATPase variants, at least two samples presented variants in genes that are part of endosomal/lysosomal/autophagosomal networks including ABCA8, ABCC6, AGAP3, ATG9A, CTSB, DNAJC13, GALC, NPC1, SLC15A3, SLC31A2, and TMEM104. CONCLUSION: Although the mechanisms involved in oral GCT initiation and progression remain unclear, our results suggest that oral GCTs have V-ATPase variants similarly to GCTs from other tissues/organs, and additionally show variants in lysosomes/endosomes/autophagosomal genes.

Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

The Y-chromosome of the Soliga, an ancient forest-dwelling tribe of South India.

A previous autosomal STR study provided evidence of a connection between the ancient Soliga tribe at the southern tip of the Indian subcontinent and Australian aboriginal populations, possibly reflecting an eastbound coastal migration circa (15 Kya). The Soliga are considered to be among India's earliest inhabitants. In this investigation, we focus on the Y chromosomal characteristics shared between the Soliga population and other Indian tribes as well as western Eurasia and Sub-Saharan Africa groups. Some noteworthy findings of this present analysis include the following: The three most frequent haplogroups detected in the Soliga population are F*, H1 and J2. F*, the oldest (43 to 63 Kya), has a significant frequency bias in favor of Indian tribes versus castes. This observation coupled with the fact that Y-STR haplotypes shared with sub-Saharan African populations are found only in F* males of the Soliga, Irula and Kurumba may indicate a unique genetic connection between these Indian tribes and sub-Saharan Africans. In addition, our study suggests that haplogroup H is confined mostly to South Asia and immediate neighbors and the H1 network may indicate minimal sharing of Y-STR haplotypes among South Asian collections, tribal and otherwise. Also, J2, brought into India by Neolithic farmers, is present at a significantly higher frequency in caste versus tribal communities. This last observation may reflect the marginalization of Indian tribes to isolated regions not ideal for agriculture.

The Y-chromosome of the Soliga, an ancient forest-dwelling tribe of South India.

A previous autosomal STR study provided evidence of a connection between the ancient Soliga tribe at the southern tip of the Indian subcontinent and Australian aboriginal populations, possibly reflecting an eastbound coastal migration circa (15 Kya). The Soliga are considered to be among India's earliest inhabitants. In this investigation, we focus on the Y chromosomal characteristics shared between the Soliga population and other Indian tribes as well as western Eurasia and Sub-Saharan Africa groups. Some noteworthy findings of this present analysis include the following: The three most frequent haplogroups detected in the Soliga population are F*, H1 and J2. F*, the oldest (43 to 63 Kya), has a significant frequency bias in favor of Indian tribes versus castes. This observation coupled with the fact that Y-STR haplotypes shared with sub-Saharan African populations are found only in F* males of the Soliga, Irula and Kurumba may indicate a unique genetic connection between these Indian tribes and sub-Saharan Africans. In addition, our study suggests that haplogroup H is confined mostly to South Asia and immediate neighbors and the H1 network may indicate minimal sharing of Y-STR haplotypes among South Asian collections, tribal and otherwise. Also, J2, brought into India by Neolithic farmers, is present at a significantly higher frequency in caste versus tribal communities. This last observation may reflect the marginalization of Indian tribes to isolated regions not ideal for agriculture.

Mutant H3 histones drive human pre-leukemic hematopoietic stem cell expansion and promote leukemic aggressiveness.

Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.

Investigating the genetic diversity and affinities of historical populations of Tibet.

This study elucidates Y chromosome distribution patterns in the three general provincial populations of historical Tibet, Amdo (n = 88), Dotoe (n = 109) and U-Tsang (n = 153) against the backdrop of 37 Asian reference populations. The central aim of this study is to investigate the genetic affinities of the three historical Tibetan populations among themselves and to neighboring populations. Y-SNP and Y-STR profiles were assessed in these historical populations. Correspondence analyses (CA) were generated with Y-SNP haplogroup data. Y-STR haplotypes were determined and employed to generate multidimensional scaling (MDS) plots based on Rst distances. Frequency contour maps of informative Y haplogroups were constructed to visualize the distributions of specific chromosome types. Network analyses based on Y-STR profiles of individuals under specific Y haplogroups were generated to examine the genetic heterogeneity among populations. Average gene diversity values and other parameters of population genetics interest were estimated to characterize the populations. The Y chromosomal results generated in this study indicate that using two sets of markers (Y-SNP, and Y-STR) the three Tibetan populations are genetically distinct. In addition, U-Tsang displays the highest gene diversity, followed by Amdo and Dotoe. The results of this transcontinental biogeographical investigation also indicate various degrees of paternal genetic affinities among these three Tibetan populations depending on the type of loci (Y-SNP or Y-STR) analyzed. The CA generated with Y-SNP haplogroup data demonstrates that Amdo and U-Tsang are closer to each other than to any neighboring non-Tibetan group. In contrast, the MDS plot based on Y-STR haplotypes displays Rst distances that are much shorter between U-Tsang and its geographic nearby populations of Ladakh, Punjab, Kathmandu and Newar than between it and Amdo. Moreover, although Dotoe is isolated from all other groups using both types of marker systems, it lies nearer to the other Tibetan collections in the Y-SNP CA than in the Y-STR MDS plot. High resolution and shallow evolutionary time frames engendered by Y-STR based analyses may reflect a more recent demographic history than that delineated by the more conserved Y-SNP markers.

Author Correction: Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

In the version of this article originally published, the main-text sentence "In three patients of European ancestry, we identified the germline variant encoding p.Ile97Met in TIM-3, which was homozygous in two (P12 and P13) and heterozygous in one (P15) in the germline but with no TIM-3 plasma membrane expression in the tumor" misstated the identifiers of the two homozygous individuals, which should have been P13 and P14. The error has been corrected in the HTML, PDF and print versions of the paper.

TRPV4 and KRAS and FGFR1 gain-of-function mutations drive giant cell lesions of the jaw.

Giant cell lesions of the jaw (GCLJ) are debilitating tumors of unknown origin with limited available therapies. Here, we analyze 58 sporadic samples using next generation or targeted sequencing and report somatic, heterozygous, gain-of-function mutations in KRAS, FGFR1, and p.M713V/I-TRPV4 in 72% (42/58) of GCLJ. TRPV4 p.M713V/I mutations are exclusive to central GCLJ and occur at a critical position adjacent to the cation permeable pore of the channel. Expression of TRPV4 mutants in HEK293 cells leads to increased cell death, as well as increased constitutive and stimulated channel activity, both of which can be prevented using TRPV4 antagonists. Furthermore, these mutations induce sustained activation of ERK1/2, indicating that their effects converge with that of KRAS and FGFR1 mutations on the activation of the MAPK pathway in GCLJ. Our data extend the spectrum of TRPV4 channelopathies and provide rationale for the use of TRPV4 and RAS/MAPK antagonists at the bedside in GCLJ.

Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

Characterizing temporal genomic heterogeneity in pediatric high-grade gliomas.

Pediatric high-grade gliomas (pHGGs) are aggressive neoplasms representing approximately 20% of brain tumors in children. Current therapies offer limited disease control, and patients have a poor prognosis. Empiric use of targeted therapy, especially at progression, is increasingly practiced despite a paucity of data regarding temporal and therapy-driven genomic evolution in pHGGs. To study the genetic landscape of pHGGs at recurrence, we performed whole exome and methylation analyses on matched primary and recurrent pHGGs from 16 patients. Tumor mutational profiles identified three distinct subgroups. Group 1 (n = 7) harbored known hotspot mutations in Histone 3 (H3) (K27M or G34V) or IDH1 (H3/IDH1 mutants) and co-occurring TP53 or ACVR1 mutations in tumor pairs across the disease course. Group 2 (n = 7), H3/IDH1 wildtype tumor pairs, harbored novel mutations in chromatin modifiers (ZMYND11, EP300 n = 2), all associated with TP53 alterations, or had BRAF V600E mutations (n = 2) conserved across tumor pairs. Group 3 included 2 tumors with NF1 germline mutations. Pairs from primary and relapsed pHGG samples clustered within the same DNA methylation subgroup. ATRX mutations were clonal and retained in H3G34V and H3/IDH1 wildtype tumors, while different genetic alterations in this gene were observed at diagnosis and recurrence in IDH1 mutant tumors. Mutations in putative drug targets (EGFR, ERBB2, PDGFRA, PI3K) were not always shared between primary and recurrence samples, indicating evolution during progression. Our findings indicate that specific key driver mutations in pHGGs are conserved at recurrence and are prime targets for therapeutic development and clinical trials (e.g. H3 post-translational modifications, IDH1, BRAF V600E). Other actionable mutations are acquired or lost, indicating that re-biopsy at recurrence will provide better guidance for effective targeted therapy of pHGGs.