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Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

Th17 cells are potent mediators in autoimmune diseases, and RORγt is required for their development. Recent studies have shown that RORγt+ Treg cells in the gut regulate intestinal inflammation by inhibiting effector T cell function. In the current study, we report that RORγt+ Treg cells were also found in lymph nodes following immunization. Not only distinct from intestinal RORγt+ Treg cells in their transcriptomes, peripheral RORγt+ Treg cells were derived from Foxp3+ thymic Treg cells in an antigen-specific manner. Development of these RORγt+ Treg cells, coined T regulatory 17 (Tr17) cells, depended on IL-6/Stat3 signaling. Tr17 cells showed suppressive activity against antigen-specific effector T cells in vitro. In addition, Tr17 cells efficiently inhibited myelin-specific Th17-cell-mediated CNS auto-inflammation in a passive EAE model. Collectively, our study demonstrates that Tr17 cells are effector Treg cells that potentially restrict autoimmunity.

TP53 is one of the most commonly mutated genes in human cancer. Mutation or loss of TP53 can occur during the early stages of oncogenesis or as a later event as tumors progress to more aggressive forms. We used a patient-derived xenograft (PDX) model of triple negative breast cancer (TNBC) to investigate how p53 deficiency contributes to metastasis. The PDX line was generated by engrafting the primary breast tumor of a patient with TP53 wild-type metastatic TNBC into the humanized mammary fat pad of NOD/SCID mice. Isogenic lines differing only in TP53 status were generated from this model by silencing p53. Here we demonstrate that the p53-proficient and -deficient tumors metastasized from the mouse mammary gland and colonized the lung, liver, bone, brain, and lymph nodes with similar frequency. p53-deficient tumors grew faster in both the primary and metastatic sites as a result of increased mitosis and decreased apoptosis. Circulating tumor cells (CTCs) are currently being evaluated in p53-proficient and –deficient PDX models. Gene expression profiling identified candidate genes with potential clinical relevance that may be responsible for increased tumor growth upon p53 loss in late stage tumors. This model is also being used to identify genes that promote breast cancer metastasis. To enrich for metastasis, metastatic subpopulations of tumor cells were isolated from lungs and serially passaged in vivo. This serial passaging enriched for metastasis to multiple secondary sites. A transcriptional signature of putative metastasis genes was generated and is being functionalized with high throughput screening in vivo. Citation Format: Powell E, Shao J, Mistry N, Yuan Y, Tieu T, Decker K, Chen H-C, Cai S, Do K-A, Heffernan TP, Edwards J, Piwnica-Worms D, Piwnica-Worms H. p53 deficiency enhances metastatic potential of triple negative breast cancer by promoting growth in primary and metastatic sites. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-08.

SummaryMyelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34+ CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

Summary Amplification of 1q21 occurs in approximately 30% of de novo and 70% of relapsed multiple myeloma (MM) and is correlated with disease progression and drug resistance. Here, we provide evidence that the 1q21 amplification-driven overexpression of ILF2 in MM promotes tolerance of genomic instability and drives resistance to DNA-damaging agents. Mechanistically, elevated ILF2 expression exerts resistance to genotoxic agents by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65, which promotes mRNA processing and the stabilization of transcripts involved in homologous recombination in response to DNA damage. The intimate link between 1q21-amplified ILF2 and the regulation of RNA splicing of DNA repair genes may be exploited to optimize the use of DNA-damaging agents in patients with high-risk MM.

SummaryA hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

Th17 cells are potent mediators in autoimmune diseases and RORγt is required for their development. Recent studies have shown that RORγt+ Treg cells in the gut regulate intestinal inflammation by inhibiting effector T cell function. In the current study, we report that RORγt+ Treg cells were also found in lymph nodes following immunization. Not only distinct from intestinal RORγt+ Treg in their transcriptomes, peripheral RORγt+ Treg cells were derived from Foxp3+ thymic Treg cells, in an antigen-specific manner. Development of these RORγt+ Treg cells, coined as T regulatory 17 (Tr17) cells, depended on IL-6/Stat3 signaling. Tr17 cells showed suppressive activity against antigen-specific effector T cells in vitro. In addition, Tr17 cells efficiently inhibited myelin-specific Th17 cell-mediated CNS auto-inflammation in a passive EAE model. Collectively, our study demonstrates Tr17 cells as effector Treg cells that potentially restrict autoimmunity.

Background Despite advances in early diagnosis and treatment of cancer patients, metastasis remains the major cause of mortality. TP53 is one of the most frequently mutated genes in human cancer, and these alterations can occur during the early stages of oncogenesis or as later events as tumors progress to more aggressive forms. Previous studies have suggested that p53 plays a role in cellular pathways that govern metastasis. To investigate how p53 deficiency contributes to late-stage tumor growth and metastasis, we developed paired isogenic patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC) differing only in p53 status for longitudinal analysis. Methods Patient-derived isogenic human tumor lines differing only in p53 status were implanted into mouse mammary glands. Tumor growth and metastasis were monitored with bioluminescence imaging, and circulating tumor cells (CTCs) were quantified by flow cytometry. RNA-Seq was performed on p53-deficient and p53 wild-type tumors, and functional validation of a lead candidate gene was performed in vivo. Results Isogenic p53 wild-type and p53-deficient tumors metastasized out of mammary glands and colonized distant sites with similar frequency. However, p53-deficient tumors metastasized earlier than p53 wild-type tumors and grew faster in both primary and metastatic sites as a result of increased proliferation and decreased apoptosis. In addition, greater numbers of CTCs were detected in the blood of mice engrafted with p53-deficient tumors. However, when normalized to tumor mass, the number of CTCs isolated from mice bearing parental and p53-deficient tumors was not significantly different. Gene expression profiling followed by functional validation identified B cell translocation gene 2 (BTG2), a downstream effector of p53, as a negative regulator of tumor growth both at primary and metastatic sites. BTG2 expression status correlated with survival of TNBC patients. Conclusions Using paired isogenic PDX-derived metastatic TNBC cells, loss of p53 promoted tumor growth and consequently increased tumor cell shedding into the blood, thus enhancing metastasis. Loss of BTG2 expression in p53-deficient tumors contributed to this metastatic potential by enhancing tumor growth in primary and metastatic sites. Furthermore, clinical data support conclusions generated from PDX models and indicate that BTG2 expression is a candidate prognostic biomarker for TNBC. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0673-9) contains supplementary material, which is available to authorized users.

Mycobacterium lepromatosis is a newly discovered cause of leprosy. Here, we present a near-complete genome of M. lepromatosis from strain FJ924 obtained from a patient who died of leprosy. The genome contained 3,215,823 nucleotides and matched ~87% with the Mycobacterium leprae genome. This genome is likely the smallest of all mycobacterial genomes known to date.

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of β2-spectrin (β2SP, encoded by SPTBN1), a SMAD adaptor for TGF-β signaling, is causally associated with BWS; however, a role of TGF-β deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-β signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-β-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-β inducible and facilitates TGF-β-mediated repression of TERT transcription via interactions with β2SP and SMAD3. This regulation was abrogated in TGF-β-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-β pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Background: Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder with a prevalence of 1 in 14,000. Its clinical manifestations include an 800-fold increased risk of tumorigenesis; yet the precise mechanism of BWS-associated tumorigenesis remains elusive. In a previous study, we showed that epigenetic silencing of β2-spectrin (β2SP, gene SPTBN1), a cytoskeletal protein and a Smad adaptor for Transforming Growth Factor-beta (TGF-β) signaling, was a causal factor in BWS. To determine the mechanism for the oncogenic switch, and whether it is related to the role of β2SP in TGF-β signaling transduction or secondary to its cytoskeletal functions, we analyzed disruption of two elements of the TGF-β pathway by generating double heterozygous Sptbn1+/−/Smad3+/− mice. Materials & Methods: Tumorigenesis analysis in Sptbn1+/− /Smad3+/− mice. Whole-transcriptome sequencing and IPA analyses were performed in the BWS cells and Sptbn1+/−; Sptbn1−/−; Smad3+/−; and Sptbn1+/−/Smad3+/− MEFs. Immunohistochemical analyses were performed for determining the expression of TERT in BWS tumors and mice liver, pancreas and stomach tissue specimens. ChIP assays were performed for examining the recruitment of β2SP/Smad3/CTCF complex at the promoter of TERT. Results: (1) 80% of Sptbn1+/−/Smad3+/− mice developed multiple tumors that were phenotypically similar to those of patients with BWS. (2) Somatic mutations in SPTBN1 and SMAD3 occur in multiple human sporadic cancers. (3) Disruption of the TGF-β pathway in Sptbn1+/−/Smad3+/− mice and BWS cells. (4) β2SP Interacts with SMAD3 in a TGF-β-dependent manner. (5) β2SP and SMAD3 interact with CTCF in the cell nucleus. (6) Dysfunction of the β2SP/SMAD3/CTCF complex increases hTERT levels in BWS and in β2SP+/-/Smad3+/- mice. Conclusions: We present a novel model of the molecular pathogenesis of BWS by demonstrating a potential shift arising from disruption of TGF-β signaling leading to BWS-associated tumorigenesis and human sporadic cancer development. Citation Format: Jian Chen, Jiun-Sheng Chen, Zhixing Yao, Wilma Jogunoori, Bibhuti Mishra, Franklin H. Herlong, Yun Seong Jeong, Nipun A. Mistry, Jianping Zhang, Xiaoping Su, Asif Rashid, Marta Davila, John R. Stroehlein, Jerry W. Shay, Lopa Mishra. Disruption of TGF-β-regulated CTCF suppression of telomerase links a human stem cell disorder to tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2334. doi:10.1158/1538-7445.AM2015-2334

Myelodysplastic syndrome (MDS) is a heterogeneous group of hematopoietic neoplastic disorders that are characterized by ineffective myeloid differentiation and dysplasia as well as telomere shortening and accumulated DNA damage in progenitor cells. Less understood is whether DNA damage is the instigator of impaired progenitor cell differentiation and MDS development. Telomerase deficient mice have served as a model system to demonstrate the adverse effects of wide-spread endogenous DNA damage signaling on stem cell function in vivo. In recent studies, we sought to determine whether persistent physiological DNA damage can impair the function of specific hematopoietic lineages by employing the 4-hydroxytamoxifen (OHT)-inducible telomerase reverse transcriptase-estrogen receptor (TERTER) model. For the first time, we demonstrate that late generation TERTER/ER mice with dysfunctional telomeres exhibit hallmark features of MDS, including peripheral blood cytopenias, bone marrow (BM) hyper-cellularity, and an increased myeloid-to-erythroid progenitor ratio in the absence of increased apoptosis. Severe tri-lineage myelodysplasia, and an increase of immature, morphologically abnormal myeloid blasts frequently with pronounced monocytic differentiation were consistent with refractory anemia with excess of blasts (RAEB) or chronic myelo-monocytic leukemia (CMML), a specific sub-group of MDS that is characterized by a high propensity to develop acute myeloid leukemia (AML). Accordingly, approximately 5% of aged TERTER/ER mice progressed to AML, as demonstrated by a marked increase of BM myeloid blasts, and infiltration of myeloid precursors into the splenic white-red pulp architecture, resulting in myeloid sarcoma with the complete effacement of lymphoid follicles. Compared to control mice with intact telomeres, the progenitor compartment of telomere dysfunctional mice shows a significant increase in the number of granulocyte-macrophage progenitors (GMP) with a concomitant loss of the megakaryocyte-erythroid progenitors (MEP) and slight reduction in the number of common myeloid progenitors (CMP), which is consistent with the condition of skewed myeloid differentiation occurring in MDS patients with higher risk of leukemic transformation. Transplantation experiments of long-term hematopoietic stem cells isolated from telomere dysfunctional mice into wild type congenic recipients revealed that the level of donor-derived skewed myeloid differentiation was comparable to that observed at steady state in the same telomere dysfunctional mice before transplantation, suggesting that impaired progenitor differentiation occurred as a result of cell intrinsic defects of telomere dysfunctional hematopoietic cells. In the setting of telomere dysfunction, somatic in vivo and in vitro telomerase reactivation reduced DNA damage signaling and specifically reversed defective differentiation and MDS phenotypes. Unbiased transcriptomic network analyses of CMP with telomere dysfunction revealed profound down-regulation of genes in the mRNA splicing and processing pathways which was rescued by telomerase reactivation, indicating that telomere dysfunction-induced DNA damage response can impact on the expression of genes involved in splicing regulation. RNA-seq analysis of telomere dysfunctional CMP suggested altered splicing activity at the level of exon usage and identified aberrantly spliced variants of genes implicated in chromatin remodeling, and histone modifications. The prominence of aberrantly spliced epigenetic regulators prompted us to hypothesize that there was a link between impaired myeloid differentiation and aberrant splicing patterns as a result of telomere dysfunction-induced repression of splicing components. In conclusion, our studies have revealed an unanticipated link between telomere biology, RNA splicing, and MDS pathogenesis and support the development of strategies designed to modulate the downstream targets of splicing alterations in specific hematopoietic populations. Disclosures No relevant conflicts of interest to declare.