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Abstract Background Microarray-based gene expression analysis of peripheral whole blood is a common strategy in the development of clinically relevant biomarker panels for a variety of human diseases. However, the results of such an analysis are often plagued by decreased sensitivity and reliability due to the effects of relatively high levels of globin mRNA in whole blood. Globin reduction assays have been shown to overcome such effects, but they require large amounts of total RNA and may induce distinct gene expression profiles. The Illumina whole genome DASL assay can detect gene expression levels using partially degraded RNA samples and has the potential to detect rare transcripts present in highly heterogeneous whole blood samples without the need for globin reduction. We assessed the utility of the whole genome DASL assay in an analysis of peripheral whole blood gene expression profiles. Results We find that gene expression detection is significantly increased with the use of whole genome DASL compared to the standard IVT-based direct hybridization. Additionally, globin-probe negative whole genome DASL did not exhibit significant improvements over globin-probe positive whole genome DASL. Globin reduction further increases the detection sensitivity and reliability of both whole genome DASL and IVT-based direct hybridization with little effect on raw intensity correlations. Raw intensity correlations between total RNA and globin reduced RNA were 0.955 for IVT-based direct hybridization and 0.979 for whole genome DASL. Conclusions Overall, the detection sensitivity of the whole genome DASL assay is higher than the IVT-based direct hybridization assay, with or without globin reduction, and should be considered in conjunction with globin reduction methods for future blood-based gene expression studies.

Motivation: Next-generation sequencing technology is transitioning quickly from research labs to clinical settings. The diagnosis and treatment selection for many acquired and autosomal conditions necessitate a method for accurately detecting somatic and germline variants., Results: We have developed Pisces, a rapid, versatile and accurate small-variant calling suite designed for somatic and germline amplicon sequencing applications. Accuracy is achieved by four distinct modules, each incorporating a number of novel algorithmic strategies., Availability and Implementation: Pisces is distributed under an open source license and can be downloaded from https://github.com/Illumina/Pisces. Pisces is available on the BaseSpace™ SequenceHub. It is distributed on Illumina sequencing platforms such as the MiSeq™ and is included in the Praxis™ Extended RAS Panel test which was recently approved by the FDA., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

This study examined whether differential DNA methylation is associated with clinical features of more aggressive disease at diagnosis and prostate cancer recurrence in African American men, who are more likely to die from prostate cancer than other populations. Tumor tissues from 76 African Americans diagnosed with prostate cancer who had radical prostatectomy as their primary treatment were profiled for epigenome-wide DNA methylation levels. Long-term follow-up identified 19 patients with prostate cancer recurrence. Twenty-three CpGs were differentially methylated (FDR q≤0.25, mean methylation difference≥0.10) in patients with vs. without recurrence, including CpGs in GCK, CDKL2, PRDM13, and ZFR2. Methylation differences were also observed between men with metastatic-lethal prostate cancer vs. no recurrence (five CpGs), regional vs. local pathological stage (two CpGs), and higher vs. lower tumor aggressiveness (one CpG). These results indicate that differentially methylated CpG sites identified in tumor tissues of African American men may contribute to prostate cancer aggressiveness., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Primary angle-closure glaucoma (PACG) is a common form of glaucoma in the Far East. Its defining feature is iridocorneal angle closure. In addition to PACG, indications of angle closure are included in the diagnostic criteria of related conditions primary angle-closure suspect (PACS) and primary angle closure (PAC). To the best of our knowledge, a causative gene for iridocorneal angle closure in humans has not been identified. This study aimed to identify the genetic cause of iridocorneal angle closure in a pedigree with at least 10 individuals diagnosed with PACS, PAC or PACG. Results of linkage analysis, segregation analysis of 44 novel variations, whole exome sequencing of 10 individuals, screenings of controls and bioinformatics predictions identified a mutation in COL18A1 that encodes collagen type XVIII as the most likely cause of angle closure in the pedigree. The role of COL18A1 in the etiology of Knobloch syndrome (KS) that is consistently accompanied by optic anomalies, available functional data on the encoded protein and the recognized role of collagens and the extracellular matrix in glaucoma pathogenesis supported the proposed role of the COL18A1 mutation in the pedigree. Subsequent identification of other COL18A1 mutations in PACS affected individuals of two unrelated families further supported that COL18A1 may affect angle closure. These PACS individuals were parents and grandparents of KS-affected children. In conclusion, a gene that affects angle closure in humans, a critical feature of PACG, has been identified. The findings also reinforce the importance of collagens in eye features and functions.

Background: Prostate cancer (PCa) with loss of the tumor suppressor gene PTEN has an unfavorable prognosis. DNA methylation profiles associated with PTEN loss may provide further insights into the mechanisms underlying these more aggressive, clinically relevant tumors., Methods: The cohort included patients with clinically localized PCa. Samples taken from the primary tumor were used to determine PTEN genomic deletions using FISH, and to analyze epigenome-wide DNA methylation profiles. Patients were followed for PCa recurrence on average for 8 years after diagnosis., Results: The study included 471 patients with data on PTEN loss, and the frequency of hemi- and homozygous PTEN loss was 10.0% and 4.5%, respectively. Loss of PTEN was associated with a significantly higher risk of recurrence (any vs. no PTEN loss; HR = 1.74; 95% CI: 1.03-2.93). Hazard ratios for hemi- and homozygous loss were 1.39 (95% CI: 0.73-2.64) and 2.84 (95% CI: 1.30-6.19), respectively. Epigenome-wide methylation profiling identified 4,208 differentially methylated CpGs (FDR Q-value < 0.01) in tumors with any versus no PTEN loss. There were no genome-wide significant differentially methylated CpGs in homo- versus hemizygous deleted tumors. Tumor methylation data were used to build a methylation signature of PTEN loss in our cohort, which was confirmed in TCGA, and included CpGs in ATP11A , GDNF , JAK1 , JAM3 , and VAPA ., Conclusion: Loss of PTEN was positively associated with PCa recurrence. Prostate tumors with PTEN loss harbor a distinct methylation signature, and these aberrantly methylated CpG sites may mediate tumor progression when PTEN is deleted., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Purpose: Aside from Gleason sum, few factors accurately identify the subset of prostate cancer patients at high risk for metastatic progression. We hypothesized that epigenetic alterations could distinguish prostate tumors with life-threatening potential., Experimental Design: Epigenome-wide DNA methylation profiling was performed in surgically resected primary tumor tissues from a population-based (n = 430) and a replication (n = 80) cohort of prostate cancer patients followed prospectively for at least 5 years. Metastasis was confirmed by positive bone scan, MRI, CT, or biopsy, and death certificates confirmed cause of death. AUC, partial AUC (pAUC, 95% specificity), and P value criteria were used to select differentially methylated CpG sites that robustly stratify patients with metastatic-lethal from nonrecurrent tumors, and which were complementary to Gleason sum., Results: Forty-two CpG biomarkers stratified patients with metastatic-lethal versus nonrecurrent prostate cancer in the discovery cohort, and eight of these CpGs replicated in the validation cohort based on a significant (P < 0.05) AUC (range, 0.66-0.75) or pAUC (range, 0.007-0.009). The biomarkers that improved discrimination of patients with metastatic-lethal prostate cancer include CpGs in five genes (ALKBH5, ATP11A, FHAD1, KLHL8, and PI15) and three intergenic regions. In the validation dataset, the AUC for Gleason sum alone (0.82) significantly increased with the addition of four individual CpGs (range, 0.86-0.89; all P <0.05)., Conclusions: Eight differentially methylated CpGs that distinguish patients with metastatic-lethal from nonrecurrent tumors were validated. These novel epigenetic biomarkers warrant further investigation as they may improve prognostic classification of patients with clinically localized prostate cancer and provide new insights on tumor aggressiveness. Clin Cancer Res; 23(1); 311-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Identifying the subset of patients with clinically localized prostate cancer (PCa) at the highest risk of recurrence remains challenging, and better prognostic markers are needed. Gleason score is the best predictor of PCa aggressiveness and prognosis. In the present study, we generated an epigenetic signature based on high versus low Gleason score tumors and evaluated its ability to predict recurrence after radical prostatectomy., Methods: Genome-wide DNA methylation data from The Cancer Genome Atlas (TCGA; no. of patients = 333) and the elastic net method were used to generate an epigenetic signature by contrasting patients with high (8-10) versus low (≤6) Gleason score tumors. The signature was then tested in a cohort of 523 patients with clinically localized disease who had radical prostatectomy. Samples taken from the primary tumor were used for DNA methylation and mRNA expression profiling. Patients were followed for PCa recurrence on average for 8 years after diagnosis., Results: The epigenetic signature includes 52 differentially methylated CpG sites. In the testing cohort, the signature was associated with poorer recurrence-free survival (hazard ratio per 25 % increase = 1.78; 95 % confidence interval 1.48, 2.16). The signature significantly improved the area under the curve (AUC) for PCa recurrence compared to clinical-pathological parameters alone, particularly among patients diagnosed with Gleason score 7 tumors (0.64 vs. 0.76, P = 1.34E-4). Results were comparable for patients with Gleason 3 + 4 and those with 4 + 3 tumors. Gene Set Enrichment Analysis showed that higher levels of the signature were associated with increased expression of genes related to cell cycle proliferation and decreased expression of androgen-responsive genes., Conclusions: This report shows evidence that DNA methylation patterns measured in prostate tumor cells are predictive of PCa aggressiveness. The epigenetic signature may have clinical utility to improve prognostication particularly in patients with intermediate Gleason score 7 tumors.

Background: DNA methylation has been hypothesized as a mechanism for explaining the association between smoking and adverse prostate cancer (PCa) outcomes. This study was aimed at assessing whether smoking is associated with prostate tumor DNA methylation and whether these alterations may explain in part the association of smoking with PCa recurrence and mortality., Methods: A total of 523 men had radical prostatectomy as their primary treatment, detailed smoking history data, long-term follow-up for PCa outcomes, and tumor tissue profiled for DNA methylation. Ninety percent of the men also had matched tumor gene expression data. A methylome-wide analysis was conducted to identify differentially methylated regions (DMRs) by smoking status. To select potential functionally relevant DMRs, their correlation with the messenger RNA (mRNA) expression of corresponding genes was evaluated. Finally, a smoking-related methylation score based on the top-ranked DMRs was created to assess its association with PCa outcomes., Results: Forty DMRs were associated with smoking status, and 10 of these were strongly correlated with mRNA expression (aldehyde oxidase 1 [AOX1], claudin 5 [CLDN5], early B-cell factor 1 [EBF1], homeobox A7 [HOXA7], lectin galactoside-binding soluble 3 [LGALS3], microtubule-associated protein τ [MAPT], protocadherin γ A [PCDHGA]/protocadherin γ B [PCDHGB], paraoxonase 3 [PON3], synaptonemal complex protein 2 like [SYCP2L], and zinc finger and SCAN domain containing 12 [ZSCAN12]). Men who were in the highest tertile for the smoking-methylation score derived from these DMRs had a higher risk of recurrence (odds ratio [OR], 2.29; 95% confidence interval [CI], 1.42-3.72) and lethal disease (OR, 4.21; 95% CI, 1.65-11.78) in comparison with men in the lower 2 tertiles., Conclusions: This integrative molecular epidemiology study supports the hypothesis that smoking-associated tumor DNA methylation changes may explain at least part of the association between smoking and adverse PCa outcomes. Future studies are warranted to confirm these findings and understand the implications for improving patient outcomes. Cancer 2016;122:2168-77. © 2016 American Cancer Society., (© 2016 American Cancer Society.)

Background: We aimed to identify the genetic cause of neurological disease in an Iranian family whose manifestations include symptoms of parkinsonism and cognitive dysfunction., Methods: Clinical data on the patients were gathered by interviews with parents, neurological examinations, and laboratory tests. Genetic analysis was performed by genome-wide single-nucleotide polymorphism homozygosity mapping and exome sequencing. The effect of putative disease-causing mutation was assessed by immunocytochemistry on HEK293 cells and Western blotting on proteins extracted from HEK293 cells transfected with wild-type and mutated genes., Results: Homozygosity mapping and exome sequencing led to identification of a mutation in ADORA1 that causes p.Gly279Ser in the encoded protein, adenosine A1 receptor (A1 R), as the probable cause of disease. The mutation segregated with disease status in the family, affects a highly conserved amino acid, and was absent in 700 controls., Conclusions: The known biological activities of A1 R in brain functions including its physical interaction with and inhibitory effect on dopamine receptor D1 provide supportive evidence that disruptions of A1 R may result in neurological dysfunction. Also, recent evidence on the related adenosine A2B receptor marks the domain in which the mutation is positioned as important for function. Finally, ADORA1 is located within the Parkinson's disease locus PARK16, which has been identified in several populations. ADORA1 may be the PD susceptibility gene within this locus. The molecular mechanism by which p.Gly279Ser disrupts A1 R function remains unknown, but a quantitative effect on interaction with the dopamine receptor was not shown. © 2016 International Parkinson and Movement Disorder Society., (© 2016 International Parkinson and Movement Disorder Society.)

The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons--GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification., (Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

We aimed to identify the genetic cause of a neurologic disorder accompanied with mental deficiency in a consanguineous family with 3 affected siblings by linkage analysis and exome sequencing. Iron accumulation in the brain of the patients was a notable phenotypic feature. A full-field electroretinography revealed generalized dysfunction of photoreceptors, bipolar cells, and amacrine cells. A splice site mutation in GTPBP2 that encodes GTP-binding protein 2 was identified in the patients and considered possible cause of their disease. The mutation was empirically shown to cause deletion of exon 9 of the gene and result in production of a truncated protein-lacking conserved C-terminus domains. GTPBP2 is a member of the GTPase superfamily of proteins. A recent report of identification of another splice site mutation in GTPBP2 in mice that causes neurodegeneration, and retinal damage provides supportive evidence for our finding. The conditions in the affected individuals of the family studied may define a novel form of neurodegeneration with brain iron accumulation, and GTPBP2 may be a novel neurodegeneration with brain iron accumulation gene., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: About half of all prostate cancers harbor the TMPRSS2:ERG (T2E) gene fusion. While T2E-positive and T2E-negative tumors represent specific molecular subtypes of prostate cancer (PCa), previous studies have not yet comprehensively investigated how these tumor subtypes differ at the epigenetic level. We therefore investigated epigenome-wide DNA methylation profiles of PCa stratified by T2E status., Results: The study included 496 patients with clinically localized PCa who had a radical prostatectomy as primary treatment for PCa. Fluorescence in situ hybridization (FISH) "break-apart" assays were used to determine tumor T2E-fusion status, which showed that 266 patients (53.6 %) had T2E-positive PCa. The study showed global DNA methylation differences between tumor subtypes. A large number of differentially methylated CpG sites were identified (false-discovery rate [FDR] Q-value <0.00001; n = 27,876) and DNA methylation profiles accurately distinguished between tumor T2E subgroups. A number of top-ranked differentially methylated CpGs in genes (FDR Q-values ≤1.53E-29) were identified: C3orf14, CACNA1D, GREM1, KLK10, NT5C, PDE4D, RAB40C, SEPT9, and TRIB2, several of which had a corresponding alteration in mRNA expression. These genes may have various roles in the pathogenesis of PCa, and the calcium-channel gene CACNA1D is a known ERG-target. Analysis of The Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings., Conclusions: This study identified substantial differences in DNA methylation profiles of T2E-positive and T2E-negative tumors, thereby providing further evidence that different underlying oncogenic pathways characterize these molecular subtypes.

Background: Aberrant DNA methylation may promote prostate carcinogenesis. We investigated epigenome-wide DNA methylation profiles in prostate cancer (PCa) compared to adjacent benign tissue to identify differentially methylated CpG sites., Methods: The study included paired PCa and adjacent benign tissue samples from 20 radical prostatectomy patients. Epigenetic profiling was done using the Infinium HumanMethylation450 BeadChip. Linear models that accounted for the paired study design and False Discovery Rate Q-values were used to evaluate differential CpG methylation. mRNA expression levels of the genes with the most differentially methylated CpG sites were analyzed., Results: In total, 2,040 differentially methylated CpG sites were identified in PCa versus adjacent benign tissue (Q-value < 0.001), the majority of which were hypermethylated (n = 1,946; 95%). DNA methylation profiles accurately distinguished between PCa and benign tissue samples. Twenty-seven top-ranked hypermethylated CpGs had a mean methylation difference of at least 40% between tissue types, which included 25 CpGs in 17 genes. Furthermore, for 10 genes over 50% of promoter region CpGs were hypermethylated in PCa versus benign tissue. The top-ranked differentially methylated genes included three genes that were associated with both promoter hypermethylation and reduced gene expression: SCGB3A1, HIF3A, and AOX1. Analysis of The Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings., Conclusions: This study of PCa versus adjacent benign tissue showed many differentially methylated CpGs and regions in and outside gene promoter regions, which may potentially be used for the development of future epigenetic-based diagnostic tests or as therapeutic targets., (© 2015 Wiley Periodicals, Inc.)

Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.

Hereditary motor and sensory neuropathy with proximal predominance (HMSN-P) is a rare disease so far identified only in individuals of Far East ancestry. Here, genome-wide linkage analysis and exome sequencing in an Iranian pedigree with 16 members affected with a neuromuscular disease led to identification of a mutation in TFG that causes p.Pro285Leu as cause of disease. The very same mutation was reported as cause of HMSN-P during the course of the study. Phenotypic analysis in conjunction with genetic data revealed that the Iranian patients were also affected with HMSN-P. Therefore, HMSN-P is not confined to the Far East and may simply not have been diagnosed in other populations. Haplotype analysis suggests at least 3 independent origins for mutated alleles that cause p.Pro285Leu. The phenotypic data gathered included subjective, biochemical, nerve conduction, electromyography, and muscle magnetic resonance imaging data. Comparison with patients with same disease in previous publications showed that clinical variability exists, sensory nerves are prominently affected, and proximal and distal muscles are involved., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Intellectual disability like other common diseases is often complex because they are genetically heterogeneous, with many different genetic defects giving rise to clinically indistinguishable phenotypes. We present diagnosis of cystathionine beta-synthase (CBS) deficiency in a multiply affected Iranian family with obvious intellectual disability based on whole genome SNP homozygosity mapping. Diagnosis based on clinical presentations had not been made because of unavailability of appropriate medical services. Genetic analysis led to identification of homozygous c.346G>A in CBS that causes p.Gly116Arg in the encoded protein, cystathionine beta-synthase. CBS is the most common causative gene of homocystinurea. Later, the same mutation was found in three other apparently unrelated Iranian homocystinuria patients. p.Gly116Arg was reported once before in a Turkish patient, suggesting it may be a common CBS deficiency causing mutation in the Middle East. Clinical features of the patients are reported that evidence to variable presentations caused by the same mutation. Finally, observations in heterozygous carriers of the mutation suggest data that a single allele of the p.Gly116Arg causing mutation may have phenotypic consequences, including cardiac related phenotypes. Our study attests to the powers of genetic analysis for diagnosis especially for some forms of intellectual disability, with known genetic causing agents., (Copyright © 2014 Elsevier B.V. All rights reserved.)

5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals at base resolution. Implementing this approach, termed "TAB-array", we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular progenitors and neural precursor cells. With the ability to discriminate 5mC and 5hmC, we identified a large number of novel dynamically methylated genomic regions that are implicated in the development of these lineages. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We present results of homozygosity mapping in two siblings affected with early onset Parkinson's disease (EOPD) and mutation screening of ATP13A2 in these and other Iranian EOPD patients. Genome-wide SNP homozygosity analysis revealed linkage to a locus that included ATP13A2, and sequencing of the gene revealed a novel p.Gln858*-causing mutation in the homozygous state in the siblings. Sequencing of the gene in seven other unrelated EOPD patients previously shown not to have mutations in PRKN, DJ-1, PINK1, and LRRK2 identified the same homozygous p. Gln858*-causing mutation in another patient. Haplotype analysis revealed that two alleles harboring the mutation were not identical by decent. The variation identified represents the 13th known disease causing mutation in ATP13A2. The clinical features of the patients who harbored the mutation are compared to those of previously reported patients with mutations in ATP13A2. Bradykinesia and rigidity, but not tremor, were reported in nearly all the patients. l-dopa administration, though initially effective, usually caused dyskinesia upon prolonged usage. Eye movement abnormalities including saccades and supranuclear gaze palsy, were almost always observed. Dystonia and bulbar anomalies were common but more variable manifestations. Although a degree of cognitive decline was found in most of the patients, the decline was often mild and absent in one patient. Age at onset of symptoms was usually in the second decade of life, and sometimes in the third decade., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: One challenge in prostate cancer is distinguishing indolent from aggressive disease at diagnosis. DNA promoter hypermethylation is a frequent epigenetic event in prostate cancer, but few studies of DNA methylation in relation to features of more aggressive tumors or prostate cancer recurrence have been completed., Methods: We used the Infinium HumanMethylation450 BeadChip to assess DNA methylation in tumor tissue from 407 patients with clinically localized prostate cancer who underwent radical prostatectomy. Recurrence status was determined by follow-up patient surveys, medical record review, and linkage with the Surveillance, Epidemiology, and End Results (SEER) registry. The methylation status of 14 genes for which promoter hypermethylation was previously correlated with advanced disease or biochemical recurrence was evaluated. Average methylation level for promoter region CpGs in patients who recurred compared with those with no evidence of recurrence was analyzed. For two genes with differential methylation, time to recurrence was examined., Results: During an average follow-up of 11.7 years, 104 (26%) patients recurred. Significant promoter hypermethylation in at least 50% of CpG sites in two genes, ABHD9 and HOXD3, was found in tumors from patients who recurred compared with those without recurrence. Evidence was strongest for HOXD3 (lowest P = 9.46 × 10(-6)), with higher average methylation across promoter region CpGs associated with reduced recurrence-free survival (P = 2 × 10(-4)). DNA methylation profiles did not differ by recurrence status for the other genes., Conclusions: These results validate the association between promoter hypermethylation of ADHB9 and HOXD3 and prostate cancer recurrence., Impact: Tumor DNA methylation profiling may help to distinguish patients with prostate cancer at higher risk for disease recurrence., (©2014 American Association for Cancer Research.)

Purpose: Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing., Methods: Two NGS assays - a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) - were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated., Results: In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were >99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays., Conclusion: This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.

We applied Illumina Human Methylation450K array to perform a genomic-scale single-site resolution DNA methylation analysis in neuronal and nonneuronal (primarily glial) nuclei separated from the orbitofrontal cortex of postmortem human brain. The findings were validated using enhanced reduced representation bisulfite sequencing. We identified thousands of sites differentially methylated (DM) between neuronal and nonneuronal cells. The DM sites were depleted within CpG-island-containing promoters but enriched in predicted enhancers. Classification of the DM sites into those undermethylated in neurons (neuronal type) and those undermethylated in nonneuronal cells (glial type), combined with findings of others that methylation within control elements typically negatively correlates with gene expression, yielded large sets of predicted neuron-specific and non-neuron-specific genes. These sets of predicted genes were in excellent agreement with the available direct measurements of gene expression in human and mouse. We also found a distinct set of DNA methylation patterns that were unique for neuronal cells. In particular, neuronal-type differential methylation was overrepresented in CpG island shores, enriched within gene bodies but not in intergenic regions, and preferentially harbored binding motifs for a distinct set of transcription factors, including neuron-specific activity-dependent factors. Finally, non-CpG methylation was substantially more prevalent in neurons than in nonneuronal cells.

Ovarian cancer remains the leading cause of death in women with gynecologic malignancies, despite surgical advances and the development of more effective chemotherapeutics. As increasing evidence indicates that clear-cell ovarian cancer may have unique pathogenesis, further understanding of molecular features may enable us to begin to understand the underlying biology and histology-specific information for improved outcomes. To study epigenetics in clear-cell ovarian cancer, fresh frozen tumor DNA (n = 485) was assayed on Illumina Infinium HumanMethylation450 BeadChips. We identified a clear-cell ovarian cancer tumor methylation profile (n = 163) which we validated in two independent replication sets (set 1, n = 163; set 2, n = 159), highlighting 22 CpG loci associated with nine genes (VWA1, FOXP1, FGFRL1, LINC00340, KCNH2, ANK1, ATXN2, NDRG21 and SLC16A11). Nearly all of the differentially methylated CpGs showed a propensity toward hypermethylation among clear-cell cases. Several loci methylation inversely correlated with tumor gene expression, most notably KCNH2 (HERG, a potassium channel) (P = 9.5 × 10(-7)), indicating epigenetic silencing. In addition, a predicted methylation class mainly represented by the clear-cell cases (20 clear cell out of 23 cases) had improved survival time. Although these analyses included only 30 clear-cell carcinomas, results suggest that loss of expression of KCNH2 (HERG) by methylation could be a good prognostic marker, given that overexpression of the potassium (K(+)) channel Eag family members promotes increased proliferation and results in poor prognosis. Validation in a bigger cohort of clear-cell tumors of the ovary is warranted.

SUCLA2 is one of several nuclear-encoded genes that can cause encephalomyopathy accompanied by mitochondrial DNA depletion. The disorder usually manifests in early childhood and leads to early death. The gene encodes one of the subunits of succinyl-CoA synthase, the enzyme that catalyzes the reversible conversion of substrates succinyl-CoA and ADP to products succinate and ATP in the tricarboxylic acid pathway. Thirty-two individuals harboring mutations in SUCLA2 have so far been reported, and five different mutations were observed among these individuals. Here we report identification of a novel mutation in SUCLA2 in two cousins affected with encephalomyopathy. The novel mutation causes p.Asp251Asn; the affected amino acid is likely positioned within the ATP-grasp domain of the encoded protein. As previously reported in other patients, we did not observe elevation of methylmalonic acid, the biochemical hallmark of patients with mutations in SUCLA2. We instead found elevated levels of succinylcarnitine.

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance.

Standard whole-genome genotyping technologies are unable to determine haplotypes. Here we describe a method for rapid and cost-effective long-range haplotyping. Genomic DNA is diluted and distributed into multiple aliquots such that each aliquot receives a fraction of a haploid copy. The DNA template in each aliquot is amplified by multiple displacement amplification, converted into barcoded sequencing libraries using Nextera technology, and sequenced in multiplexed pools. To assess the performance of our method, we combined two male genomic DNA samples at equal ratios, resulting in a sample with diploid X chromosomes with known haplotypes. Pools of the multiplexed sequencing libraries were subjected to targeted pull-down of a 1-Mb contiguous region of the X-chromosome Duchenne muscular dystrophy gene. We were able to phase the Duchenne muscular dystrophy region into two contiguous haplotype blocks with a mean length of 494 kb. The haplotypes showed 99% agreement with the consensus base calls made by sequencing the individual DNAs. We subsequently used the strategy to haplotype two human genomes. Standard genomic sequencing to identify all heterozygous SNPs in the sample was combined with dilution-amplification-based sequencing data to resolve the phase of identified heterozygous SNPs. Using this procedure, we were able to phase >95% of the heterozygous SNPs from the diploid sequence data. The N50 for a Yoruba male DNA was 702 kb whereas the N50 for a European female DNA was 358 kb. Therefore, the strategy described here is suitable for haplotyping of a set of targeted regions as well as of the entire genome.

Background & Aims: Cholangiocarcinoma, the second most common liver cancer, can be classified as intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma. We performed an integrative genomic analysis of ICC samples from a large series of patients., Methods: We performed a gene expression profile, high-density single-nucleotide polymorphism array, and mutation analyses using formalin-fixed ICC samples from 149 patients. Associations with clinicopathologic traits and patient outcomes were examined for 119 cases. Class discovery was based on a non-negative matrix factorization algorithm and significant copy number variations were identified by Genomic Identification of Significant Targets in Cancer (GISTIC) analysis. Gene set enrichment analysis was used to identify signaling pathways activated in specific molecular classes of tumors, and to analyze their genomic overlap with hepatocellular carcinoma (HCC)., Results: We identified 2 main biological classes of ICC. The inflammation class (38% of ICCs) is characterized by activation of inflammatory signaling pathways, overexpression of cytokines, and STAT3 activation. The proliferation class (62%) is characterized by activation of oncogenic signaling pathways (including RAS, mitogen-activated protein kinase, and MET), DNA amplifications at 11q13.2, deletions at 14q22.1, mutations in KRAS and BRAF, and gene expression signatures previously associated with poor outcomes for patients with HCC. Copy number variation-based clustering was able to refine these molecular groups further. We identified high-level amplifications in 5 regions, including 1p13 (9%) and 11q13.2 (4%), and several focal deletions, such as 9p21.3 (18%) and 14q22.1 (12% in coding regions for the SAV1 tumor suppressor). In a complementary approach, we identified a gene expression signature that was associated with reduced survival times of patients with ICC; this signature was enriched in the proliferation class (P < .001)., Conclusions: We used an integrative genomic analysis to identify 2 classes of ICC. The proliferation class has specific copy number alterations, activation of oncogenic pathways, and is associated with worse outcome. Different classes of ICC, based on molecular features, therefore might require different treatment approaches., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Neurodegeneration with brain iron accumulation (NBIA) constitutes a group of neurodegenerative disorders with pronounced iron deposition in the basal ganglia. PANK2 mutations are the most common cause of these disorders. C19orf12 was recently reported as another causative gene. We present phenotypic data and results of screening of PANK2 and C19orf12 in 11 unrelated Iranian NBIA patients., Methods: Phenotypic data were obtained by neurologic examination, magnetic resonance imaging, and interviews. Mutation screening of PANK2 and C19orf12 was performed by sequencing., Results: PANK2 and C19orf12 mutations were found in 7 and 4 patients, respectively. Phenotypic comparisons suggest that C19orf12 mutations as compared with PANK2 mutations result in a milder disease course., Conclusions: Mutations in both PANK2 and C19orf12 contributed significantly to NBIA in the Iranian patients. To the best of our knowledge, this is the first genetic analysis reported on a cohort of NBIA patients from the Middle East., (Copyright © 2012 Movement Disorders Society.)

The ability to measure human aging from molecular profiles has practical implications in many fields, including disease prevention and treatment, forensics, and extension of life. Although chronological age has been linked to changes in DNA methylation, the methylome has not yet been used to measure and compare human aging rates. Here, we build a quantitative model of aging using measurements at more than 450,000 CpG markers from the whole blood of 656 human individuals, aged 19 to 101. This model measures the rate at which an individual's methylome ages, which we show is impacted by gender and genetic variants. We also show that differences in aging rates help explain epigenetic drift and are reflected in the transcriptome. Moreover, we show how our aging model is upheld in other human tissues and reveals an advanced aging rate in tumor tissue. Our model highlights specific components of the aging process and provides a quantitative readout for studying the role of methylation in age-related disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have extensively characterized the DNA methylomes of 139 patients with chronic lymphocytic leukemia (CLL) with mutated or unmutated IGHV and of several mature B-cell subpopulations through the use of whole-genome bisulfite sequencing and high-density microarrays. The two molecular subtypes of CLL have differing DNA methylomes that seem to represent epigenetic imprints from distinct normal B-cell subpopulations. DNA hypomethylation in the gene body, targeting mostly enhancer sites, was the most frequent difference between naive and memory B cells and between the two molecular subtypes of CLL and normal B cells. Although DNA methylation and gene expression were poorly correlated, we identified gene-body CpG dinucleotides whose methylation was positively or negatively associated with expression. We have also recognized a DNA methylation signature that distinguishes new clinico-biological subtypes of CLL. We propose an epigenomic scenario in which differential methylation in the gene body may have functional and clinical implications in leukemogenesis.

Latent transforming growth factor (TGF) beta-binding protein 2 (LTBP2) is an extracellular matrix (ECM) protein that associates with fibrillin-1 containing microfibrils. Various factors prompted considering LTBP2 in the etiology of isolated ectopia lentis and associated conditions such as Weill-Marchesani syndrome (WMS) and Marfan syndrome (MFS). LTBP2 was screened in 30 unrelated Iranian patients. Mutations were found only in one WMS proband and one MFS proband. Homozygous c.3529G>A (p.Val1177Met) was shown to cause autosomal recessive WMS or WM-like syndrome by several approaches, including homozygosity mapping. Light, fluorescent, and electron microscopy evidenced disruptions of the microfibrillar network in the ECM of the proband's skin. In conjunction with recent findings regarding other ECM proteins, the results presented strongly support the contention that anomalies in WMS patients are due to disruptions in the ECM. Heterozygous c.1642C >T (p.Arg548*) possibly contributed to MFS-related phenotypes, including ocular manifestations, mitral valve prolapse, and pectus excavatum, but was not cause of MFS., (© 2012 Wiley Periodicals, Inc.)

Ichthyosis is a heterogeneous disorder characterized by abnormal skin scaling over the whole body. Autosomal recessive congenital ichthyosis (ARCI) comprises various forms, the most important of which are lamellar ichthyosis (LI) and congenital ichthyosiform erythroderma (CIE). Seven genes have been identified to be causative of ARCI, and these account for disease in 60-80% of the patients. There is notable phenotypic overlap between the major forms of ARCI, and a strong genotype-phenotype correlation has not been found. Here, we initially aimed to identify the causative gene in a large Iranian ARCI pedigree, and subsequently performed genetic analysis on four other affected pedigrees. A genotype-phenotype correlation was sought. Whole genome homozygosity mapping using high-density single nucleotide polymorphism chips was performed on the large pedigree. Linkage to chromosome 5 and a mutation in NIPAL4 causing p.G297R were identified. The same mutation was also identified in two of the remaining four Iranian pedigrees. Two of the NIPAL4 mutation bearing pedigrees were classified as CIE and one as LI. Notably, all NIPAL4 mutation-bearing patients manifested diffuse yellowish keratoderma on the palms and soles. We provide evidence suggesting presentation of this diffuse yellowish keratoderma may be indicative of mutations in NIPAL4, providing an easily assessable genotype-phenotype correlation., (© 2011 Japanese Dermatological Association.)

Background: We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs., Methodology/principal Findings: The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input., Conclusions/significance: In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology.

Many mammalian genes are occupied by paused RNA polymerase II (pol II) in the promoter-proximal region on both sides of the transcription start site. However, the impact of pol II pausing on gene expression and cell biology is not fully understood. In this study, we used a Cre-Lox system to conditionally knock out the b subunit of mouse negative elongation factor (Nelf-b), a key pol II-pausing factor, in mouse embryonic fibroblasts. We found that Nelf-b was associated with the promoter-proximal region of the majority of expressed genes, yet genetic ablation of Nelf-b only affected the steady-state mRNA levels of a small percentage of the Nelf-b-associated genes. Interestingly, Nelf-b deletion also increased levels of transcription start site upstream transcripts at multiple negative elongation factor-associated genes. The direct target genes of Nelf-b were highly enriched with those involved in the control of cell growth and cell death. Correspondingly, Nelf-b knock-out mouse embryonic fibroblasts exhibited slower progression from quiescence to proliferation, as well as in a cycling cell population. Furthermore, Nelf-b deletion also resulted in increased apoptosis. Thus, the genetic and genomic studies provide new physiological and molecular insight into Nelf-mediated pol II pausing.

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R2 of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: We have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens., Methodology/principal Findings: We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng)., Conclusions/significance: Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).

Objective: Despite recent advances in the conceptual understanding of the pathogenesis of ovarian cancer, it remains the foremost cause of death from gynecologic malignancies in developed countries. The main reason for such a high rate of mortality is the lack of sensitive and specific biomarkers and imaging techniques for early detection of ovarian cancer. Additional biological insights into early-stage ovarian carcinogenesis are needed to help speed the development of markers for early detection of ovarian cancer. The objective of this study was to characterize differentially expressed genes in high-grade stage I serous carcinoma of the ovary., Methods: We analyzed gene expression in macrodissected formalin-fixed, paraffin-embedded samples from 5 high-grade stage I serous carcinomas and 5 stage I borderline tumors of the ovary using the Illumina Whole Genome DASL assay (cDNA-mediated annealing, selection, extension, and ligation) corresponding to 24,000 genes. Significance Analysis of Microarrays was performed to determine differentially expressed genes in stage I serous carcinoma, and class prediction analysis was performed to determine the predictive value of differentially expressed gene sets to correctly classify serous carcinoma from borderline tumors in 3 independent data sets. Altered transcription factor pathways and biological pathways unique to stage I serous carcinoma were identified through class comparison of differentially expressed genes., Results: Unsupervised cluster analysis of gene expression correctly classified stage I serous carcinomas from serous borderline tumors. Supervised analysis identified several known, as well as novel, genes differentially expressed in stage I ovarian cancer. Using a differentially expressed gene set, class comparison prediction analysis correctly identified serous carcinomas from serous borderline tumors in 3 independent data sets at over 80% accuracy, sensitivity, and specificity. Pathway analysis demonstrated the significance of p53 and E2F pathways in serous carcinogenesis and significant involvements of cell cycle and immune response pathways in stage I serous epithelial ovarian cancer., Conclusion: We have identified differentially expressed genes associated with the carcinogenesis of high-grade stage I serous EOC. Furthermore, integrative analysis of biological and transcription pathway data contributed to the confirmation of important biological pathways and discovery of additional unique genes and pathways that may have potential importance in ovarian pathogenesis and biomarker development.