To evaluate the utility of preoperative multiparametric magnetic resonance imaging (MP-MRI) in predicting biochemical recurrence (BCR) following radical prostatectomy (RP).

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

Oncocytomas are mostly benign tumors characterized by accumulation of defective mitochondria, and in sporadic cases, are associated with disruptive mitochondrial DNA (mtDNA) mutations. However, the role mtDNA mutations have in renal tumors of Birt-Hogg-Dubé (BHD) patients and other renal oncocytomas with an apparent genetic component has not been investigated to date. Here we characterize the mitochondrial genome in different renal tumors and investigate the possibility of employing mtDNA sequencing analyses of biopsy specimens to aid in the differential diagnosis of oncocytomas. The entire mitochondrial genome was sequenced in 25 samples of bilateral and multifocal (BMF) renal oncocytomas, 30 renal tumors from BHD patients and 36 non-oncocytic renal tumors of different histologies as well as in biopsy samples of kidney tumors. mtDNA sequencing in BMF oncocytomas revealed that all tumors carry disruptive mutations, which impair the assembly of the NADH-ubiquinone oxidoreductase. Multiple tumors from a given BMF oncocytoma patient mainly harbor the same somatic mutation and the kidneys of these patients display diffuse oncocytosis. In contrast, renal oncocytomas of patients with BHD syndrome and renal tumors with different histologies do not show disruptive mtDNA mutations. Moreover, we demonstrate that it is feasible to amplify and sequence the entire mtDNA in biopsy specimens, and that these sequences are representative of the tumor DNA. These results show that pathogenic mtDNA mutations affecting complex I of the respiratory chain are strongly correlated with the oncocytoma phenotype in non-BHD-related renal tumors and that mtDNA sequences from biopsies are predictive of the tumor genotype. This work supports a role for mtDNA mutations in respiratory chain complexes as diagnostic markers for renal oncocytomas.

The utility of induced pluripotent stem cells (iPSCs) as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. Here we report the whole-genome sequencing of three human iPSC lines derived from two cell types of an adult donor by episomal vectors. The vector sequence was undetectable in the deeply sequenced iPSC lines. We identified 1,058-1,808 heterozygous single-nucleotide variants (SNVs), but no copy-number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but ~50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during integration-free iPSC induction.

Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.

Micrognathia, glossoptosis, and cleft palate comprise one of the most common malformation sequences, Robin sequence. It is a component of the TARP syndrome, talipes equinovarus, atrial septal defect, Robin sequence, and persistent left superior vena cava. This disorder is X-linked and severe, with apparently 100% pre- or postnatal lethality in affected males. Here we characterize a second family with TARP syndrome, confirm linkage to Xp11.23-q13.3, perform massively parallel sequencing of X chromosome exons, filter the results via a number of criteria including the linkage region, use a unique algorithm to characterize sequence changes, and show that TARP syndrome is caused by mutations in the RBM10 gene, which encodes RNA binding motif 10. We further show that this previously uncharacterized gene is expressed in midgestation mouse embryos in the branchial arches and limbs, consistent with the human phenotype. We conclude that massively parallel sequencing is useful to characterize large candidate linkage intervals and that it can be used successfully to allow identification of disease-causing gene mutations.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Despite the tremendous drop in the cost of nucleotide sequencing in recent years, many research projects still utilize sequencing of pools containing multiple samples for the detection of sequence variants as a cost saving measure. Various software tools exist to analyze these pooled sequence data, yet little has been reported on the relative accuracy and ease of use of these different programs.

There are conflicting reports on whether familial nonmedullary thyroid cancer is more aggressive than sporadic nonmedullary thyroid cancer. Our aim was to determine if the clinical and pathologic characteristics of familial nonmedullary thyroid cancer are different than nonmedullary thyroid cancer.

Hypertension occurs at a higher rate in African Americans than in European Americans. Based on the assumption that causal variants are more frequently found on DNA segments inherited from the ancestral population with higher disease risk, we employed admixture mapping to identify genetic loci with excess local African ancestry associated with blood pressure. Chromosomal regions 1q21.2-21.3, 4p15.1, 19q12 and 20p13 were significantly associated with diastolic blood pressure (β = 5.28, -7.94, -6.82 and 5.89, P-value = 6.39E-04, 2.07E-04, 6.56E-05 and 5.04E-04, respectively); 1q21.2-21.3 and 19q12 were also significantly associated with mean arterial pressure (β = 5.86 and -6.40, P-value = 5.32E-04 and 6.37E-04, respectively). We further selected SNPs that had large allele frequency differences within these regions and tested their association with blood pressure. SNP rs4815428 was significantly associated with diastolic blood pressure after Bonferroni correction (β = -2.42, P-value = 9.57E-04), and it partially explained the admixture mapping signal at 20p13. SNPs rs771205 (β = -1.99, P-value = 3.37E-03), rs3126067, rs2184953 and rs58001094 (the latter three exhibit strong linkage disequilibrium, β = -2.3, P-value = 1.4E-03) were identified to be significantly associated with mean arterial pressure, and together they fully explained the admixture signal at 1q21.2-21.3. Although no SNP at 4p15.1 showed large ancestral allele frequency differences in our dataset, we detected association at low-frequency African-specific variants that mapped predominantly to the gene PCDH7, which is most highly expressed in aorta. Our results suggest that these regions may harbor genetic variants that contribute to the different prevalence of hypertension.

MicroRNAs (miRNAs) are small, non-coding RNA molecules with multiple roles in many biological processes. Few studies have shown the molecular characteristics in younger prostate cancer (PCa) patients. In this study, we performed miRNA profiling in young PCa (EO-PCa) cases compared with PCa arising in older men (LO-PCa). Experimental Design: Formalin-fixed, paraffin embedded tissue was used. miRNA was extracted for PCR array and NanoString methods. Relative miRNAs expression levels were obtained by comparing young vs older men, and young PCa tumor samples vs normal epithelium. Results: miRNA profiling showed a different expression pattern in PCa arising in younger men, and young PCa tumoral and its normal counterpart. Nine miRNAs (hsa-miR-140-5p, hsa-miR-146a, hsa-miR-29b, hsa-miR-9, hsa-miR-124-3p, hsa-let-7f-5p, hsa-miR-184, hsa-miR-373, hsa-miR-146b-5p) showed differences in the expression compared to LO-PCa. Fourteen miRNAs were significantly up-regulated (miR-1973, miR-663a, miR-575, miR-93-5p, miR-630, miR-600, miR-494, miR-150-5p, miR-137, miR-25-3p, miR-375, miR-489, miR-888-5p, miR-142-3p), while 9 were found down-regulated (miR-21-5p, miR-363-3p, miR-205-5p, miR-548ai, miR-3195, 145-5p, miR-143-3p, miR-222-3p, miR-221-3p) comparing young PCa tumoral tissue compared to normal counterpart. The higher expression of miR-600 and miR-137 were associated with high Gleason score, extraprostatic extension and lymphatic invasion. Conclusion: These results suggest that PCa in younger patients has a different expression profile compared to normal tissue and PCa arising in older man. Differentially expressed miRNAs provide insights of molecular mechanisms involve in this PCa subtype.

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.

Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt-Hogg-Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.

In this study, we aimed to evaluate the protein expression profile of a spectrum of renal cell carcinomas (RCC) to find potential biomarkers for disease onset and progression and therefore, prospective therapeutic targets. A 2D-gel based proteomic analysis was used to outline differences in protein levels among different subtypes of renal cell carcinomas, including clear cell carcinomas, papillary lesions, chromophobe tumors and renal oncocytomas. Spot pattern was compared to the corresponding normal kidney from the same patients and distinctive, differentially expressed proteins were characterized by mass spectrometry. Twenty-one protein spots were found differentially expressed between clear cell RCC and normal tissue and 38 spots were found expressed in chromophobe tumors. Eleven proteins were identified, with most differentially expressed -by fold change- between clear cell tumors and the corresponding normal tissue. Two of the identified proteins, Triosephosphate isomerase 1 (TPI-1) and Heat Shock protein 27 (Hsp27), were further validated in a separate set of tumors by immunohistochemistry and expression levels were correlated with clinicopathologic features of the patients. Hsp27 was highly expressed in 82% of the tumors used for validation, and all cases showed strong immunoreactivity for TPI-1. In both Hsp27 and TPI-1, protein expression positively correlated with histologic features of the disease. Our results suggest that the subjacent cytogenetic abnormalities seen in different histological types of RCC are followed by specific changes in protein expression. From these changes, Hsp27 and TPI-1 emerged as potential candidates for the differentiation and prognosis in RCC.

In order to understand the role of miRNAs in renal tumorigenesis, we undertook a stepwise approach that included a comprehensive differential miRNA expression analysis for the most common histological subtypes of human renal neoplasms appearing in either sporadic or hereditary forms. We also aimed to test the hypothesis that microRNAs can act as an alternative mechanism of VHL gene inactivation and therefore might be correlated with tumorigenesis in ccRCC. Finally, we wanted to explore whether the well-known hypoxic activation of ccRCC is followed by a specific pattern of miRNA expression.

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.

Checkpoint inhibitors have not been effective for prostate cancer as single agents. Durvalumab is a human IgG1-K monoclonal antibody that targets programmed death ligand 1 and is approved by the U.S. Food and Drug Administration for locally advanced or metastatic urothelial cancer and locally advanced, unresectable stage 3 non-small cell lung cancer. Olaparib, a poly (ADP-ribose) polymerase inhibitor, has demonstrated an improvement in median progression-free survival (PFS) in select patients with metastatic castration-resistant prostate cancer (mCRPC). Data from other trials suggest there may be improved activity in men with DNA damage repair (DDR) mutations treated with checkpoint inhibitors. This trial evaluated durvalumab and olaparib in patients with mCRPC with and without somatic or germline DDR mutations.

Neurofibromatosis type 2 (NF2) is an autosomal dominant Mendelian tumor predisposition disorder caused by germline pathogenic variants in the tumor suppressor NF2. Meningiomas are the second most common neoplasm in NF2, often occurring in multiple intracranial and spinal locations within the same patient. In this prospective longitudinal study, we assessed volumes and growth rates of ten spinal and ten cranial benign meningiomas in seven NF2 patients that concluded with surgical resection and performed whole-exome sequencing and copy-number variant (CNV) analysis of the tumors. Our comparison of the volume and the growth rate of NF2-associated spinal and cranial meningiomas point to the differences in timing of tumor initiation and/or to the differences in tumor progression (e.g., non-linear, saltatory growth) at these two anatomical locations. Genomic investigation of these tumors revealed that somatic inactivation of NF2 is the principal and perhaps the only driver of tumor initiation; and that tumor progression likely occurs via accumulation of CNVs, rather than point mutations. Results of this study contribute to a better understanding of NF2-associated meningiomas clinical behavior and their genetic underpinnings.

Immunotherapy is reportedly an effective form of therapy for some advanced cancers such as lung adenocarcinoma, malignant melanoma and colorectal adenocarcinoma. In renal cell carcinoma (RCC), the role of immunotherapy is under investigation. Programmed Death-Ligand 1 (PD-L1) is a molecule expressed on the surface of certain tumor cells and binds to the Programmed cell death protein 1 (PD-1) on cytotoxic T-cells, an interaction that inhibits the antitumor immune response. The aim of this study is to evaluate PD-L1 expression in the morphologic spectrum of RCC. A total of 172 cases of RCC comprising all types were studied and the PD-L1 was correlated with immune response for CD4 and CD8. Positive membranous staining for PD-L1 was seen in 59 (34%) of the 172 samples. The positive cases were HLRCC (31/53), Type 1 Papillary RCC (10/31), Chromophobe (7/20), Hybrid (3/9), TFE-3 related cancer (3/8), Undifferentiated (3/5), and TFEB tumors (2/2). Clear cell carcinomas, Oncocytomas and SDHB deficient-RCC didn't show any expression of PD-L1; (0/34;0/7;0/3). Our results demonstrated that aggressive forms of RCC such as HLRCC have high expression of PD-L1, in contrast to clear cell renal carcinomas. Our findings support a possible role of anti-PD-L1/PD-1 immunotherapies in the treatment of PD-L1-positive RCC.

MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies.