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1.  The Effect of Changes in Stimulus Level on Electrically Evoked Cortical Auditory Potentials 
Ear and hearing  2009;30(3):320-329.
The purpose of this study was to determine whether the electrically evoked acoustic change complex (EACC) could be used to assess sensitivity to changes in stimulus level in cochlear implant (CI) recipients and to investigate the relationship between EACC amplitude and rate of growth of the N1-P2 onset response with increases in stimulus level.
Twelve postlingually deafened adults using Nucleus CI24 CIs participated in this study. Nucleus Implant Communicator (NIC) routines were used to bypass the speech processor and to control the stimulation of the implant directly. The stimulus consisted of an 800 msec burst of a 1000 pps biphasic pulse train. A change in the stimulus level was introduced 400 msec after stimulus onset. Band-pass filtering (1 to 100 Hz) was used to minimize stimulus artifact. Four to six recordings of 50 sweeps were obtained for each condition, and averaged responses were analyzed in the time domain using standard peak picking procedures.
Cortical auditory change potentials were recorded from CI users in response to both increases and decreases in stimulation level. The amplitude of the EACC was found to be dependent on the magnitude of the stimulus change. Increases in stimulus level elicited more robust EACC responses than decreases in stimulus level. Also, EACC amplitudes were significantly correlated with the slope of the growth of the onset response.
This work describes the effect of change in stimulus level on electrically evoked auditory change potentials in CI users. The amplitude of the EACC was found to be related both to the magnitude of the stimulus change introduced and to the rate of growth of the N1-P2 onset response. To the extent that the EACC reflects processing of stimulus change, it could potentially be a valuable tool for assessing neural processing of the kinds of stimulation patterns produced by a CI. Further studies are needed, however, to determine the relationships between the EACC and psychophysical measures of intensity discrimination in CI recipients.
PMCID: PMC2896017  PMID: 19322089
2.  Effects of Long-Term Use of a Cochlear Implant on the Electrically Evoked Compound Action Potential 
Since the early 1990s, it has been possible to measure electrically evoked compound action potentials (ECAPs) from Nucleus cochlear implant users. Recording the ECAP does not require active participation by the subject, and the recordings are not adversely affected by attention or sleep, making this response an ideal tool for monitoring long-term changes. Previous research from our laboratory (Hughes et al, 2001) has shown that ECAP thresholds and slope of the ECAP growth functions are relatively stable over time. However, this conclusion was based on results obtained from a fairly limited number of study participants, each of whom used the Nucleus CI24M cochlear implant and were followed for less than two years.
To evaluate the effect of long-term use of a cochlear implant on ECAP thresholds and slope of the ECAP input/output function for both pediatric and adult cochlear implant recipients.
Research Design
A longitudinal study that describes how ECAP thresholds and growth functions change over a period of 96 mo following initial activation. Changes over time in ECAP threshold and slope of the ECAP growth function were analyzed, and effects of the subject’s age, type of CI (cochlear implant), and stimulating electrode are included in the analysis.
Study Sample
134 Nucleus CI users participated in this study. All were profoundly deaf. This subject pool included 84 individuals (40 adults and 44 children) who used the Nucleus CI24M cochlear implant and 50 individuals (21 adults and 29 children) who used the Nucleus CI24R cochlear implant.
Data Collection and Analysis
Electrodes 5, 10, 15, and 20 were stimulated, and ECAP growth functions were measured for each subject at regular intervals following the initial activation of the device.
Small increases in mean ECAP thresholds were observed for both pediatric and adult CI users between an “early” visit that occurred within 3–6 mo following hookup and a “late” visit that occurred 4.8–6 yr later. For adults, the average increase in ECAP threshold was 3.94 CL (clinical programming units for Nucleus CIs). For children, the average increase was 4.16 CL. These differences, while small, were statistically significant. Slope of the ECAP growth functions measured over the same time interval did not change significantly. On average, pediatric CI users had ECAP thresholds that were 4–5 CL units higher than the adult CI recipients. The most striking outcome from this study, however, was the finding that when compared with postlingually deafened adults, pediatric CI users had ECAP growth functions that were substantially steeper. The differences between the results obtained from children and those obtained from adults were statistically significant and largely independent of device type or stimulating electrode.
Results from this study show ECAP thresholds and growth functions to change very little over a 5–6 yr observation interval suggesting that long-term use of a CI is not likely to have a significant negative impact on the response of the peripheral auditory system. Pediatric CI users were shown to have, on average, higher ECAP thresholds and steeper ECAP growth functions than postlingually deafened adult CI users. This finding suggests potential differences between the two patient populations either in terms of the current fields within the cochlea or the effective distance between the stimulating electrode and the stimulable neural tissue.
PMCID: PMC2881552  PMID: 20085195
Auditory evoked potential; cochlear implant; compound action potential; electrical stimulation; neural response telemetry
3.  The Electrically Evoked Auditory Change Complex: Preliminary Results from Nucleus Cochlear Implant Users 
Ear and hearing  2008;29(5):704-717.
The purpose of this study was to determine if changes in the position of the stimulating electrode in the cochlea could be used to elicit the electrically evoked auditory change complex (EACC) from Nucleus cochlear implant users.
Nine postlingually deafened adults participated in this study. Each study participant had been using his or her Nucleus CI24 cochlear implant for at least 3 months prior to testing. The speech processor was bypassed and the output of the implanted receiver/stimulator was controlled directly. The stimulus was a 600 ms burst of a biphasic pulse train (1000 pps). In control conditions, the stimulating electrode was held constant and stimulation continued throughout the 600 ms recording interval. In experimental conditions, the EACC was elicited by introducing a change in the stimulating electrode 300 ms after the onset of the pulse train. The EACC was recorded using surface electrodes. Three recordings of 100 sweeps each were obtained for each stimulus condition. Bandpass filtering (1 – 100 Hz) was used to minimize contamination of the recordings by stimulus artifact. Averaged responses were then smoothed using a 40 ms wide boxcar filter and standard peak picking procedures were used to analyze these responses in the time domain.
In each case, a clear onset response (P1-N1-P2) was recorded. In the experimental conditions, a second evoked potential, the EACC, was also recorded following the change in stimulating electrode. This second response had general morphologic characteristics that were very similar to those of the onset response. Increasing the separation between the two stimulating electrodes in the experimental conditions resulted in a general trend toward increased EACC amplitudes.
This report describes results of a set of experiments in which the speech processor of the cochlear implant was bypassed and the EACC was recorded in response to a change in stimulating electrode position. EACC amplitude was shown to increase as the separation between the two stimulating electrodes increased. While preliminary in nature, these results demonstrate the feasibility of recording the EACC in response to changes in stimulating electrode position from individual cochlear implant users.
PMCID: PMC2767236  PMID: 18596644
auditory evoked potential; cochlear implant; cortical evoked potential; electrical stimulation
4.  Unique somatic and malignant expression patterns implicate PIWI-interacting RNAs in cancer-type specific biology 
Scientific Reports  2015;5:10423.
Human PIWI-interacting RNAs (piRNAs) are known to be expressed in germline cells, functionally silencing LINEs and SINEs. Their expression patterns in somatic tissues are largely uncharted. We analyzed 6,260 human piRNA transcriptomes derived from non-malignant and tumour tissues from 11 organs. We discovered that only 273 of the 20,831 known piRNAs are expressed in somatic non-malignant tissues. However, expression patterns of these piRNAs were able to distinguish tissue-of-origin. A total of 522 piRNAs are expressed in corresponding tumour tissues, largely distinguishing tumour from non-malignant tissues in a cancer-type specific manner. Most expressed piRNAs mapped to known transcripts, contrary to “piRNA clusters” reported in germline cells. We showed that piRNA expression can delineate clinical features, such as histological subgroups, disease stages, and survival. PiRNAs common to many cancer types might represent a core gene-set that facilitates cancer growth, while piRNAs unique to individual cancer types likely contribute to cancer-specific biology.
PMCID: PMC4444957  PMID: 26013764
5.  DNA Methylation Is Globally Disrupted and Associated with Expression Changes in Chronic Obstructive Pulmonary Disease Small Airways 
DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illumina’s Infinium HM27 and Affymetrix’s Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2–related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.
PMCID: PMC4068945  PMID: 24298892
chronic obstructive pulmonary disease; small airways; epigenetic regulation; DNA methylation; integrative omics
6.  A skewed view of X chromosome inactivation 
X chromosome inactivation involves a random choice to silence either X chromosome early in mammalian female development. Once silenced the inactive X is stably inherited through subsequent somatic cell divisions, and thus, females are generally mosaics, having a mixture of cells with one or the other parental X active. While in most females the number of cells with either X being active is roughly equal, skewing of X chromosome inactivation is observed in a percentage of women. In this issue of the JCI, Bolduc and colleagues address whether skewing of X chromosome inactivation in humans is influenced by an X-linked locus that can alter this initial random inactivation (see the related article beginning on page 333). Their data indicate that most of the skewing observed in humans results from secondary events rather than being due to an inherited tendency to inactivate a particular X chromosome.
PMCID: PMC2147673  PMID: 18097476
7.  Landscape of DNA methylation on the X chromosome reflects CpG density, functional chromatin state and X-chromosome inactivation 
Human Molecular Genetics  2014;24(6):1528-1539.
X-chromosome inactivation (XCI) achieves dosage compensation between males and females through the silencing of the majority of genes on one of the female X chromosomes. Thus, the female X chromosomes provide a unique opportunity to study euchromatin and heterochromatin of allelic regions within the same nuclear environment. We examined the interplay of DNA methylation (DNAm) with CpG density, transcriptional activity and chromatin state at genes on the X chromosome using over 1800 female samples analysed with the Illumina Infinium Human Methylation450 BeadChip. DNAm was used to predict an inactivation status for 63 novel transcription start sites (TSSs) across 27 tissues. There was high concordance of inactivation status across tissues, with 62% of TSSs subject to XCI in all 27 tissues examined, whereas 9% escaped from XCI in all tissues, and the remainder showed variable escape from XCI between females in subsets of tissues. Inter-female and twin data supported a model of predominately cis-acting influences on inactivation status. The level of expression from the inactive X relative to the active X correlated with the amount of female promoter DNAm to a threshold of ∼30%, beyond which genes were consistently subject to inactivation. The inactive X showed lower DNAm than the active X at intragenic and intergenic regions for genes subject to XCI, but not at genes that escape from inactivation. Our categorization of genes that escape from X inactivation provides candidates for sex-specific differences in disease.
PMCID: PMC4381753  PMID: 25381334
8.  Effects of Stimulation Level and Electrode Pairing on The Binaural Interaction Component of The Electrically Evoked Auditory Brain Stem Response 
Ear and hearing  2010;31(4):457-470.
The purpose of this study was to investigate the effects of stimulation level and electrode pairing on the binaural interaction component (BIC) of the electrically evoked auditory brain stem response (EABR) in Nucleus cochlear implant users.
Ten postlingually deafened adult cochlear implant users participated in this study. EABRs were measured using loudness balanced, biphasic current pulses presented in the left monaural, right monaural and bilateral stimulation conditions. BICs were computed based on measures of the EABR obtained for each subject by pairing the electrode 12 (out of 22 intracochlear electrodes) in the right ear with each of 11 electrodes spaced across the electrode array in the left ear. The effect of stimulation level on the amplitude of the BIC was investigated by measuring growth functions of the BIC from six subjects. The effect of electrode pairing on the amplitude of the BIC was studied at high stimulation levels in ten subjects and at low stimulation levels in seven subjects. The high stimulation level was chosen as the 90% point of the subject’s dynamic range (DR) or the highest stimulation level where the electrophysiological recordings were not contaminated by muscle artifacts. The low stimulation level was chosen as a level that was 10% point of subject’s DR higher than the BIC threshold for six of these seven subjects. For one subject, BIC thresholds were not available and the low stimulation level was referred to the 70% point of her DR.
BICs were successfully recorded from all 11 interaural electrode pairs for a majority of subjects tested at both stimulation levels. BIC amplitudes increased with stimulation level. The effect of stimulation level on latencies of the BIC was less robust. At high stimulation levels, BIC amplitudes did not change significantly as the stimulating electrode used in the left ear was systematically varied. When low stimulation levels were used, BIC amplitude was maximal for interaural electrode pairs with similar intracochlear positions and decreased when the offset between interaural electrodes increased.
This study demonstrates that stimulation level affects amplitudes of the BIC response. It is possible to record the BIC of the EABR in bilateral CI users even from interaural electrode pairs that have large interaural offsets. This finding suggests that when high-level stimuli are used, there is a broad pattern of current spread within the two cochleae. At lower stimulation levels the spread of excitation within the cochlea is reduced making the effect of electrode pairing on the amplitude of the BIC more pronounced.
PMCID: PMC4193499  PMID: 20418771
auditory evoked potential; bilateral cochlear implantation; auditory brain stem response; electrical stimulation
9.  Differentially methylated CpG island within human XIST mediates alternative P2 transcription and YY1 binding 
BMC Genetics  2014;15:89.
X-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome, the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates, triggering silencing of the chromosome. In mouse, an alternative Xist promoter, P2 is also the site of YY1 binding, which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation, including absence of a functional antisense regulator Tsix, and absence of strictly paternal inactivation in extraembryonic tissues, prompting us to examine regulatory regions for the human XIST gene.
We demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However, YY1 binding is insufficient to drive P2 expression or establish the DHS, which may require a development-specific factor. Furthermore, reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.
The differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST.
PMCID: PMC4363909  PMID: 25200388
XIST; X-chromosome inactivation; Alternative promoter; YY1; DNA methylation; Long non-coding RNA; RNA FISH; DNase hypersensitivity site
10.  Variable escape from X-chromosome inactivation: Identifying factors that tip the scales towards expression 
Bioessays  2014;36(8):746-756.
In humans over 15% of X-linked genes have been shown to ‘escape’ from X-chromosome inactivation (XCI): they continue to be expressed to some extent from the inactive X chromosome. Mono-allelic expression is anticipated within a cell for genes subject to XCI, but random XCI usually results in expression of both alleles in a cell population. Using a study of allelic expression from cultured lymphoblasts and fibroblasts, many of which showed substantial skewing of XCI, we recently reported that the expression of genes lies on a contiunuum between those that are subject to inactivation, and those that escape. We now review allelic expression studies from mouse, and discuss the variability in escape seen in both humans and mice in genic expression levels, between X chromosomes and between tissues. We also discuss current knowledge of the heterochromatic features, DNA elements and three-dimensional topology of the inactive X that contribute to the balance of expression from the otherwise inactive X chromosome.
PMCID: PMC4143967  PMID: 24913292
allelic imbalance; boundary elements; dosage compensation; epigenetic marks; RNA-seq; waystations; XIST
11.  Direct Recordings from the Auditory Cortex in a Cochlear Implant User 
Electrical stimulation of the auditory nerve with a cochlear implant (CI) is the method of choice for treatment of severe-to-profound hearing loss. Understanding how the human auditory cortex responds to CI stimulation is important for advances in stimulation paradigms and rehabilitation strategies. In this study, auditory cortical responses to CI stimulation were recorded intracranially in a neurosurgical patient to examine directly the functional organization of the auditory cortex and compare the findings with those obtained in normal-hearing subjects. The subject was a bilateral CI user with a 20-year history of deafness and refractory epilepsy. As part of the epilepsy treatment, a subdural grid electrode was implanted over the left temporal lobe. Pure tones, click trains, sinusoidal amplitude-modulated noise, and speech were presented via the auxiliary input of the right CI speech processor. Additional experiments were conducted with bilateral CI stimulation. Auditory event-related changes in cortical activity, characterized by the averaged evoked potential and event-related band power, were localized to posterolateral superior temporal gyrus. Responses were stable across recording sessions and were abolished under general anesthesia. Response latency decreased and magnitude increased with increasing stimulus level. More apical intracochlear stimulation yielded the largest responses. Cortical evoked potentials were phase-locked to the temporal modulations of periodic stimuli and speech utterances. Bilateral electrical stimulation resulted in minimal artifact contamination. This study demonstrates the feasibility of intracranial electrophysiological recordings of responses to CI stimulation in a human subject, shows that cortical response properties may be similar to those obtained in normal-hearing individuals, and provides a basis for future comparisons with extracranial recordings.
PMCID: PMC3642273  PMID: 23519390
averaged evoked potential; cortical plasticity; electrical stimulation; electrocorticography; high gamma; intracranial electrophysiology
12.  Translating Dosage Compensation to Trisomy 21 
Nature  2013;500(7462):10.1038/nature12394.
Down syndrome (DS) is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21 (Chr21). We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST. Using genome editing with zinc finger nucleases, we targeted a large, inducible XIST transgene into the Chr21 DYRK1A locus, in DS pluripotent stem cells. XIST RNA coats Chr21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a “Chr21 Barr Body.” This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Remarkably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one Chr21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of “chromosome therapy”.
PMCID: PMC3848249  PMID: 23863942
13.  Analysis of expressed SNPs identifies variable extents of expression from the human inactive X chromosome 
Genome Biology  2013;14(11):R122.
X-chromosome inactivation (XCI) results in the silencing of most genes on one X chromosome, yielding mono-allelic expression in individual cells. However, random XCI results in expression of both alleles in most females. Allelic imbalances have been used genome-wide to detect mono-allelically expressed genes. Analysis of X-linked allelic imbalance in females with skewed XCI offers the opportunity to identify genes that escape XCI with bi-allelic expression in contrast to those with mono-allelic expression and which are therefore subject to XCI.
We determine XCI status for 409 genes, all of which have at least five informative females in our dataset. The majority of genes are subject to XCI and genes that escape from XCI show a continuum of expression from the inactive X. Inactive X expression corresponds to differences in the level of histone modification detected by allelic imbalance after chromatin immunoprecipitation. Differences in XCI between populations and between cell lines derived from different tissues are observed.
We demonstrate that allelic imbalance can be used to determine an inactivation status for X-linked genes, even without completely non-random XCI. There is a range of expression from the inactive X. Genes escaping XCI, including those that do so in only a subset of females, cluster together, demonstrating that XCI and location on the X chromosome are related. In addition to revealing mechanisms involved in cis-gene regulation, determining which genes escape XCI can expand our understanding of the contributions of X-linked genes to sexual dimorphism.
PMCID: PMC4053723  PMID: 24176135
14.  Spread of X-chromosome inactivation into autosomal sequences: role for DNA elements, chromatin features and chromosomal domains 
Human Molecular Genetics  2013;23(5):1211-1223.
X-chromosome inactivation results in dosage equivalence between the X chromosome in males and females; however, over 15% of human X-linked genes escape silencing and these genes are enriched on the evolutionarily younger short arm of the X chromosome. The spread of inactivation onto translocated autosomal material allows the study of inactivation without the confounding evolutionary history of the X chromosome. The heterogeneity and reduced extent of silencing on autosomes are evidence for the importance of DNA elements underlying the spread of silencing. We have assessed DNA methylation in six unbalanced X-autosome translocations using the Illumina Infinium HumanMethylation450 array. Two to 42% of translocated autosomal genes showed this mark of silencing, with the highest degree of inactivation observed for trisomic autosomal regions. Generally, the extent of silencing was greatest close to the translocation breakpoint; however, silencing was detected well over 100 kb into the autosomal DNA. Alu elements were found to be enriched at autosomal genes that escaped from inactivation while L1s were enriched at subject genes. In cells without the translocation, there was enrichment of heterochromatic features such as EZH2 and H3K27me3 for those genes that become silenced when translocated, suggesting that underlying chromatin structure predisposes genes towards silencing. Additionally, the analysis of topological domains indicated physical clustering of autosomal genes of common inactivation status. Overall, our analysis indicated a complex interaction between DNA sequence, chromatin features and the three-dimensional structure of the chromosome.
PMCID: PMC4051349  PMID: 24158853
15.  XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells 
The establishment of facultative heterochromatin by X-chromosome inactivation requires the long non-coding RNA XIST/Xist. However, the molecular mechanism by which the RNA achieves chromosome-wide gene silencing remains unknown. Mouse Xist has been shown to have redundant domains for cis-localization, and requires a series of well-conserved tandem ‘A’ repeats for silencing. We previously described a human inducible XIST transgene that is capable of cis-localization and suppressing a downstream reporter gene in somatic cells, and have now leveraged these cells to dissect the sequences critical for XIST-dependent gene silencing in humans.
We demonstrated that expression of the inducible full-length XIST cDNA was able to suppress expression of two nearby reporter genes as well as endogenous genes up to 3 MB from the integration site. An inducible construct containing the repeat A region of XIST alone could silence the flanking reporter genes but not the more distal endogenous genes. Reporter gene silencing could also be accomplished by a synthetic construct consisting of nine copies of a consensus repeat A sequence, consistent with previous studies in mice. Progressively shorter constructs showed a linear relationship between the repeat number and the silencing capacity of the RNA. Constructs containing only two repeat A units were still able to partially silence the reporter genes and could thus be used for site-directed mutagenesis to demonstrate that sequences within the two palindromic cores of the repeat are essential for silencing, and that it is likely the first palindrome sequence folds to form a hairpin, consistent with compensatory mutations observed in eutherian sequences.
Silencing of adjacent reporter genes can be effected by as little as 94 bp of XIST, including two ‘monomers’ of the A repeat. This region includes a pair of essential palindromic sequences that are evolutionarily well-conserved and the first of these is likely to form an intra-repeat hairpin structure. Additional sequences are required for the spread of silencing to endogenous genes on the chromosome.
PMCID: PMC3734131  PMID: 23915978
X-chromosome inactivation; XIST; Long non-coding RNA; Eutherian dosage compensation; Gene silencing
16.  Additional annotation enhances potential for biologically-relevant analysis of the Illumina Infinium HumanMethylation450 BeadChip array 
Measurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potential physiologically-relevant epigenetic changes. Illumina Infinium (Illumina, San Diego, CA, USA) is a commercially available microarray suite used to measure DNAm at many sites throughout the genome. However, it has been suggested that a subset of array probes may give misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest Infinium array, the HumanMethylation450 BeadChip (450 k), with >485,000 probes covering 99% of Reference Sequence (RefSeq) genes (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA). Annotation that was added or expanded on includes: 1) documented SNPs in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites.
Probes with documented SNPs at the target CpG (4.3% of probes) were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG demonstrated how sample genotype can confound the measurement of DNAm. Additionally, 8.6% of probes mapped to multiple locations in silico. Measurements from these non-specific probes likely represent a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by an alternative high-density and intermediate-density CpG island classification provided a distinctive pattern of DNAm. Finally, variable enrichment for differentially methylated probes was noted across CpG classes and gene feature groups, dependant on the tissues that were compared.
DNAm arrays offer a high-throughput approach for which careful consideration of probe content should be utilized to better understand the biological processes affected. Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450 k array. Additionally, probe classification by CpG enrichment classes and to a lesser extent gene feature groups resulted in distinct patterns of DNAm. Thus, we recommend that compromised probes be removed from analyses and that the genomic context of DNAm is considered in studies deciphering the biological meaning of Illumina 450 k array data.
PMCID: PMC3740789  PMID: 23452981
Infinium HumanMethylation450 BeadChip array; DNA methylation; non-specific probes; Polymorphic probes; CpG islands; Annotation; CpG enrichment; Tissue-specific DNA methylation; Repetitive elements; 450 k
17.  Preliminary results of the relationship between the binaural interaction component of the electrically evoked auditory brainstem response and interaural pitch comparisons in bilateral cochlear implant recipients 
Ear and hearing  2012;33(1):57-68.
The purpose of this study was to investigate the relationship between electrophysiologic measures of the binaural interaction component (BIC) of the electrically evoked auditory brainstem response (EABR) and psychophysical measures of interaural pitch comparisons in Nucleus bilateral cochlear implant users.
Data were collected for ten postlingually deafened adult cochlear implant users. Each subject conducted an interaural pitch-comparison task using a biphasic pulse train with a pulse rate of 1000 pulses per second (pps) at high stimulation levels. Stimuli were presented in a two-interval, two-alternative forced-choice procedure with roving current variations. A subgroup of four subjects repeated the task at low stimulation levels. BICs were measured using loudness balanced, biphasic current pulses presented at a rate of 19.9 pps for each subject by pairing the electrode 12 (out of 22 intracochlear electrodes) in the right ear with each of 11 electrodes spaced across the electrode array in the left ear. The BIC was measured at high stimulation levels in ten subjects and at low stimulation levels in seven subjects. Because of differences in stimulation rate used in BIC measures and interaural pitch comparisons, the actual stimulation levels were different in these two measures. The relationship between BIC responses and results of interaural pitch comparisons was evaluated for each of the individual subjects as well as at the group level. Evaluation was carried out separately for results obtained at high and low stimulation levels.
There was no significant correlation between results of BIC measures and interaural pitch comparisons on either the individual or group levels. Lower stimulation level did not improve the relationship between these two measures.
No significant correlations between psychophysical measures of interaural pitch comparisons and electrophysiologic measures of the BIC of the EABR were found. The lack of correlation may be attributed to methods used to quantify the data, small number of subjects retested at low stimulation levels, as well as central processing components involved in the interaural pitch-comparison task.
PMCID: PMC3193893  PMID: 21730858
auditory evoked potential; bilateral cochlear implantation; auditory brain stem response; electrical stimulation
18.  Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing 
Genetics  2012;192(4):1281-1293.
Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.
PMCID: PMC3512139  PMID: 23023002
X chromosome inactivation (XCI); DNA methylation; human transgenes; targeted transgenesis; Xist deletion
19.  Human Cancer Long Non-Coding RNA Transcriptomes 
PLoS ONE  2011;6(10):e25915.
Once thought to be a part of the ‘dark matter’ of the genome, long non-coding RNAs (lncRNAs) are emerging as an integral functional component of the mammalian transcriptome. LncRNAs are a novel class of mRNA-like transcripts which, despite no known protein-coding potential, demonstrate a wide range of structural and functional roles in cellular biology. However, the magnitude of the contribution of lncRNA expression to normal human tissues and cancers has not been investigated in a comprehensive manner. In this study, we compiled 272 human serial analysis of gene expression (SAGE) libraries to delineate lncRNA transcription patterns across a broad spectrum of normal human tissues and cancers. Using a novel lncRNA discovery pipeline we parsed over 24 million SAGE tags and report lncRNA expression profiles across a panel of 26 different normal human tissues and 19 human cancers. Our findings show extensive, tissue-specific lncRNA expression in normal tissues and highly aberrant lncRNA expression in human cancers. Here, we present a first generation atlas for lncRNA profiling in cancer.
PMCID: PMC3185064  PMID: 21991387
20.  The Relationship between Electrically Evoked Compound Action Potential and Speech Perception : A Study in Cochlear Implant Users with Short Electrode Array 
To determine the extent to which electrically evoked compound action potential (ECAP) measurements were related with speech perception performance in implant users with a short electrode array and to investigate the relationship between ECAP measures and performance according to specific devices.
Prospective study.
Tertiary referral center.
Seventeen Hybrid cochlear implant users were tested in this study. Subjects were divided into 2 groups: 1) 8 using the Nucleus Hybrid M, 2) 9 using the Nucleus Hybrid RE. In addition, 21 Nucleus Freedom long electrode implant (CI24RE) users were also tested to compare with the results of the old device (CI24M).
Main Outcome measures
ECAP growth functions were recorded using either an interphase gap (IPG) of 8 or 45 us. We then calculated 1) the slope of the growth function, and 2) changes in sensitivity with IPG. For each subject, these measures were compared with performance on tests of word recognition.
The changes in sensitivity using two IPGs showed no correlation with results of word recognition test in Hybrid cochlear implant users. In contrast, relatively strong correlations have been found between the slope of ECAP growth functions and performance on word recognition test. Additionally, when we separate the results of Hybrid M and RE, the slopes of ECAP growth functions from only Hybrid RE CI recipients were significantly correlated with speech performance. The slopes of ECAP growth function in CI24RE users with long electrode were also significantly correlated with performance. However, comparing between two independent correlations in RE devices, correlation was higher in Hybrid RE group.
The results presented in this paper support the view that slope of the ECAP growth can show significant correlation to performance with a cochlear implant. Further, these results suggest that the strength of the correlation may be related to the specific device. These results suggest that ECAP measures may be useful in developing a test to predict outcomes with the implant.
PMCID: PMC2933654  PMID: 20634770
electrically evoked compound action potential; cochlear implant; Hybrid electrodes
21.  Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation 
Human Genetics  2011;130(2):187-201.
X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E−16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset of genes predicted different XCI statuses in different tissues. Using previously published expression data the effect of transcription on gene-body methylation was investigated and while X-linked introns of highly expressed genes were more methylated than the introns of lowly expressed genes, exonic methylation did not differ based on expression level. We conclude that the XCI status predicted using methylation of X-linked promoters with CpG islands was usually the same as determined by expression analysis and that 12% of X-linked genes examined show tissue-specific XCI whereby a gene has a different XCI status in at least one of the four tissues examined.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-011-1007-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3132437  PMID: 21597963
22.  The functional role of long non-coding RNA in human carcinomas 
Molecular Cancer  2011;10:38.
Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer paradigm demonstrating potential roles in both oncogenic and tumor suppressive pathways. These novel genes are frequently aberrantly expressed in a variety of human cancers, however the biological functions of the vast majority remain unknown. Recently, evidence has begun to accumulate describing the molecular mechanisms by which these RNA species function, providing insight into the functional roles they may play in tumorigenesis. In this review, we highlight the emerging functional role of lncRNAs in human cancer.
PMCID: PMC3098824  PMID: 21489289
23.  Methylated DNA Immunoprecipitation 
The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.
PMCID: PMC2763296  PMID: 19241501
24.  Comparison of Electrically Evoked Compound Action Potential Thresholds and Loudness Estimates for the Stimuli Used to Program the Advanced Bionics Cochlear Implant 
In the mid-1990s, Cochlear Corporation introduced a cochlear implant (CI) to the market that was equipped with hardware that made it possible to record electrically evoked compound action potentials (ECAPs) from CI users of all ages. Over the course of the next decade, many studies were published that compared ECAP thresholds with levels used to program the speech processor of the Nucleus CI. In 2001 Advanced Bionics Corporation introduced the Clarion CII cochlear implant (the Clarion CII internal device is also known as the CII Bionic Ear). This cochlear implant was also equipped with a system that allowed measurement of the ECAP. While a great deal is known about how ECAP thresholds compare with the levels used to program the speech processor of the Nucleus CI, relatively few studies have reported comparisons between ECAP thresholds and the levels used to program the speech processor of the Advanced Bionics CI.
To explore the relationship between ECAP thresholds and behavioral measures of perceptual dynamic range for the range of stimuli commonly used to program the speech processor of the Advanced Bionics CI.
Research Design
This prospective and experimental study uses correlational and descriptive statistics to define the relationship between ECAP thresholds and perceptual dynamic range measures.
Study Sample
Twelve postlingually deafened adults participated in this study. All were experienced users of the Advanced Bionics CI system.
Data Collection and Analysis
ECAP thresholds were recorded using the commercially available SoundWave software. Perceptual measures of threshold (T-level), most comfortable level (M-level), and maximum comfortable level (C-level) were obtained using both “tone bursts” and “speech bursts.” The relationship between these perceptual and electrophysiological variables was defined using paired t-tests as well as correlation and linear regression.
ECAP thresholds were significantly correlated with the perceptual dynamic range measures studied; however, correlations were not strong. Analysis of the individual data revealed considerable discrepancy between the contour of ECAP threshold versus electrode function and the behavioral loudness estimates used for programming.
ECAP thresholds recorded from Advanced Bionics cochlear implant users always indicated levels where the programming stimulus was audible for the listener. However, the correlation between ECAP thresholds and M-levels (the primary metric used to program the speech processor of the Advanced Bionics CI), while statistically significant, was quite modest. If programming levels are to be determined on the basis of ECAP thresholds, care should be taken to ensure that stimulation is not uncomfortably loud, particularly on the basal electrodes in the array.
PMCID: PMC2875933  PMID: 20085196
Auditory evoked potential; cochlear implant; compound action potential; electrical stimulation; neural response imaging
25.  Active Chromatin Marks Are Retained on X Chromosomes Lacking Gene or Repeat Silencing Despite XIST/Xist Expression in Somatic Cell Hybrids 
PLoS ONE  2010;5(5):e10787.
X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.
Methodology/Principal Findings
To assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.
Loss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.
PMCID: PMC2875404  PMID: 20520737

Results 1-25 (31)