Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

Nuclear-embedded mitochondrial DNA sequences in 66,083 human genomes.

DNA transfer from cytoplasmic organelles to the cell nucleus is a legacy of the endosymbiotic event-the majority of nuclear-mitochondrial segments (NUMTs) are thought to be ancient, preceding human speciation1-3. Here we analyse whole-genome sequences from 66,083 people-including 12,509 people with cancer-and demonstrate the ongoing transfer of mitochondrial DNA into the nucleus, contributing to a complex NUMT landscape. More than 99% of individuals had at least one of 1,637 different NUMTs, with 1 in 8 individuals having an ultra-rare NUMT that is present in less than 0.1% of the population. More than 90% of the extant NUMTs that we evaluated inserted into the nuclear genome after humans diverged from apes. Once embedded, the sequences were no longer under the evolutionary constraint seen within the mitochondrion, and NUMT-specific mutations had a different mutational signature to mitochondrial DNA. De novo NUMTs were observed in the germline once in every 104 births and once in every 103 cancers. NUMTs preferentially involved non-coding mitochondrial DNA, linking transcription and replication to their origin, with nuclear insertion involving multiple mechanisms including double-strand break repair associated with PR domain zinc-finger protein 9 (PRDM9) binding. The frequency of tumour-specific NUMTs differed between cancers, including a probably causal insertion in a myxoid liposarcoma. We found evidence of selection against NUMTs on the basis of size and genomic location, shaping a highly heterogenous and dynamic human NUMT landscape.

An ancestral 10-bp repeat expansion in VWA1 causes recessive hereditary motor neuropathy.

The extracellular matrix comprises a network of macromolecules such as collagens, proteoglycans and glycoproteins. VWA1 (von Willebrand factor A domain containing 1) encodes a component of the extracellular matrix that interacts with perlecan/collagen VI, appears to be involved in stabilizing extracellular matrix structures, and demonstrates high expression levels in tibial nerve. Vwa1-deficient mice manifest with abnormal peripheral nerve structure/function; however, VWA1 variants have not previously been associated with human disease. By interrogating the genome sequences of 74 180 individuals from the 100K Genomes Project in combination with international gene-matching efforts and targeted sequencing, we identified 17 individuals from 15 families with an autosomal-recessive, non-length dependent, hereditary motor neuropathy and rare biallelic variants in VWA1. A single disease-associated allele p.(G25Rfs*74), a 10-bp repeat expansion, was observed in 14/15 families and was homozygous in 10/15. Given an allele frequency in European populations approaching 1/1000, the seven unrelated homozygote individuals ascertained from the 100K Genomes Project represents a substantial enrichment above expected. Haplotype analysis identified a shared 220 kb region suggesting that this founder mutation arose >7000 years ago. A wide age-range of patients (6-83 years) helped delineate the clinical phenotype over time. The commonest disease presentation in the cohort was an early-onset (mean 2.0 ± 1.4 years) non-length-dependent axonal hereditary motor neuropathy, confirmed on electrophysiology, which will have to be differentiated from other predominantly or pure motor neuropathies and neuronopathies. Because of slow disease progression, ambulation was largely preserved. Neurophysiology, muscle histopathology, and muscle MRI findings typically revealed clear neurogenic changes with single isolated cases displaying additional myopathic process. We speculate that a few findings of myopathic changes might be secondary to chronic denervation rather than indicating an additional myopathic disease process. Duplex reverse transcription polymerase chain reaction and immunoblotting using patient fibroblasts revealed that the founder allele results in partial nonsense mediated decay and an absence of detectable protein. CRISPR and morpholino vwa1 modelling in zebrafish demonstrated reductions in motor neuron axonal growth, synaptic formation in the skeletal muscles and locomotive behaviour. In summary, we estimate that biallelic variants in VWA1 may be responsible for up to 1% of unexplained hereditary motor neuropathy cases in Europeans. The detailed clinical characterization provided here will facilitate targeted testing on suitable patient cohorts. This novel disease gene may have previously evaded detection because of high GC content, consequential low coverage and computational difficulties associated with robustly detecting repeat-expansions. Reviewing previously unsolved exomes using lower QC filters may generate further diagnoses.

Human MAF1 targets and represses active RNA polymerase III genes by preventing recruitment rather than inducing long-term transcriptional arrest.

RNA polymerase III (Pol III) is tightly controlled in response to environmental cues, yet a genomic-scale picture of Pol III regulation and the role played by its repressor MAF1 is lacking. Here, we describe genome-wide studies in human fibroblasts that reveal a dynamic and gene-specific adaptation of Pol III recruitment to extracellular signals in an mTORC1-dependent manner. Repression of Pol III recruitment and transcription are tightly linked to MAF1, which selectively localizes at Pol III loci, even under serum-replete conditions, and increasingly targets transcribing Pol III in response to serum starvation. Combining Pol III binding profiles with EU-labeling and high-throughput sequencing of newly synthesized small RNAs, we show that Pol III occupancy closely reflects ongoing transcription. Our results exclude the long-term, unproductive arrest of Pol III on the DNA as a major regulatory mechanism and identify previously uncharacterized, differential coordination in Pol III binding and transcription under different growth conditions.

tRNA biology in the omics era: Stress signalling dynamics and cancer progression.

Recent years have seen a burst in the number of studies investigating tRNA biology. With the transition from a gene-centred to a genome-centred perspective, tRNAs and other RNA polymerase III transcripts surfaced as active regulators of normal cell physiology and disease. Novel strategies removing some of the hurdles that prevent quantitative tRNA profiling revealed that the differential exploitation of the tRNA pool critically affects the ability of the cell to balance protein homeostasis during normal and stress conditions. Furthermore, growing evidence indicates that the adaptation of tRNA synthesis to cellular dynamics can influence translation and mRNA stability to drive carcinogenesis and other pathological disorders. This review explores the contribution given by genomics, transcriptomics and epitranscriptomics to the discovery of emerging tRNA functions, and gives insights into some of the technical challenges that still limit our understanding of the RNA polymerase III transcriptional machinery.

Intracortical inhibitory and excitatory circuits in subjects with minimal hepatic encephalopathy: a TMS study.

Minimal hepatic encephalopathy (MHE) is the earliest form of hepatic encephalopathy (HE) and affects up to 80 % of patients with liver cirrhosis. By definition, MHE is characterized by psychomotor slowing and subtle cognitive deficits,  but obvious clinical manifestations are lacking. Given its covert nature, MHE is often underdiagnosed. This study was aimed at detecting neurophysiological changes, as assessed by means of transcranial magnetic stimulation (TMS), involved in the early pathogenesis of the HE. We investigated motor cortex excitability in 15 patients with MHE and in 15 age-matched age-matched cirrhotic patients without MHE; the resting motor threshold, the short-interval intracortical inhibition (SICI) and the intracortical facilitation (ICF) were examined. Paired-pulse TMS revealed significant increased SICI and reduced ICF in the patients with MHE. These findings may reflect abnormalities in intrinsic brain activity and altered organization of functional connectivity networks. In particular, the results suggest a shift in the balance between intracortical inhibitory and excitatory mechanisms towards a net increase of inhibitory neurotransmission. Together with other neurophysiological (in particular EEG) and neuroimaging techniques, TMS may thus provide early markers of cerebral dysfunction in cirrhotic patients with MHE.

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

MOTIVATION: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. RESULTS: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. AVAILABILITY: The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter

Noninvasive Spinal Cord Stimulation: Technical Aspects and Therapeutic Applications.

BACKGROUND: Electrical and magnetic trans-spinal stimulation can be used to increase the motor output of multiple spinal segments and modulate cortico-spinal excitability. The application of direct current through the scalp as well as repetitive transcranial magnetic stimulation are known to influence brain excitability, and hence can also modulate other central nervous system structures, including spinal cord. OBJECTIVE: This study aimed to evaluate the effects and the therapeutic usefulness of these noninvasive neuromodulatory techniques in healthy subjects and in the neurorehabilitation of patients with spinal cord disorders, as well as to discuss the possible mechanisms of action. A comprehensive review that summarizes previous studies using noninvasive spinal cord stimulation is lacking. METHODS: PubMed (MEDLINE) and EMBASE were systematically searched to identify the most relevant published studies. We performed here an extensive review in this field. RESULTS: By decreasing the spinal reflex excitability, electrical and magnetic trans-spinal stimulation could be helpful in normalizing reflex hyperexcitability and treating hypertonia in subjects with lesions to upper motor neurons. Transcutaneous spinal direct current stimulation, based on applying direct current through the skin, influences the ascending and descending spinal pathways as well as spinal reflex excitability, and there is increasing evidence that it also can induce prolonged functional neuroplastic changes. When delivered repetitively, magnetic stimulation could also modulate spinal cord functions; however, at present only a few studies have documented spastic-reducing effects induced by repetitive spinal magnetic stimulation. Moreover, paired peripheral and transcranial stimulation can be used to target the spinal cord and may have potential for neuromodulation in spinal cord-injured subjects. CONCLUSIONS: Noninvasive electrical and magnetic spinal stimulation may provide reliable means to characterize important neurophysiologic and pathophysiologic aspects of spinal cord function. Moreover, transcutaneous direct current stimulation and repetitive magnetic stimulation may hold therapeutic promise in patients with spinal cord disorders, although future well-controlled studies are needed to corroborate and extend the preliminary findings.

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III.

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.

Involvement of central sensory pathways in subjects with restless legs syndrome: A neurophysiological study.

In patients with restless legs syndrome (RLS) a motor cortical disinhibition has been reported in transcranial magnetic stimulation (TMS) studies, but the neuronal excitability in other cortical areas has been poorly explored. The aim of this study was the functional evaluation of thalamo-cortical circuits and inhibitory cortical responses in the sensory cortex in RLS. We assessed the high-frequency somatosensory evoked potentials (HF-SEP) in sixteen subjects suffering from RLS of different degrees of severity. In patients with severe or very severe RLS we found a significant desynchronization with amplitude reduction of both pre- and post-synaptic HF-SEP bursts, which suggest an impairment in the thalamo-cortical projections and in the cortical inhibitory interneurons activity, respectively. The assessment of the central sensory pathways by means of HF-SEP may shed light on the pathophysiological mechanisms of RLS.

Disinhibition of sensory cortex in patients with amyotrophic lateral sclerosis.

In patients with amyotrophic lateral sclerosis (ALS) a motor cortical hyperexcitability has been reported in transcranial magnetic stimulation studies, but little is known about the neuronal excitability in other cortical areas. The aim of the present study was the functional evaluation of the sensory cortex in subjects with ALS by assessing the high-frequency somatosensory evoked potentials (HF-SEP). No significant HF-SEP abnormalities were observed in ALS patients with disease duration of <2 years, while the patients with a disease duration of>2 years we found a large amplitude reduction of post-synaptic HF-SEP burst. Since post-synaptic burst of HF-SEP is thought to reflect the activity of cortical inhibitory interneurons, our findings provide further evidence that disinhibition is a primary characteristic of ALS that also involves the somatosensory cortex.

Ipsilateral motor evoked potentials in a patient with unihemispheric cortical atrophy due to Rasmussen encephalitis.

The role of the ipsilaterally descending motor pathways in the recovery mechanisms after unilateral hemispheric damage is still poorly understood. Motor output reorganization was investigated in a 56-year-old male patient with acquired unilateral hemispheric atrophy due to Rasmussen encephalitis. In particular, the ipsilateral corticospinal pathways were explored using focal transcranial magnetic stimulation. In the first dorsal interosseous and wrist extensors muscles, the median amplitudes of the ipsilateral motor evoked potentials induced by transcranial magnetic stimulation in the patient were higher than those of 10 age-matched healthy control subjects. In the biceps brachii muscle, the median amplitudes of the ipsilateral motor evoked potentials were the second largest in the patient compared to the controls. This study demonstrated a reinforcement of ipsilateral motor projections from the unaffected motor cortex to the hemiparetic hand in a subject with acquired unihemispheric cortical damage.

Ocular ultrasound for monitoring pseudotumor cerebri syndrome.

The aim of this study was to evaluate the feasibility of ocular ultrasound in the follow-up of pseudotumor cerebri syndrome and to correlate ultrasound with clinical parameters. In a cohort study of 22 consecutive adult patients, ocular ultrasound was performed measuring optic nerve sheath diameter, optic nerve diameter, and optic disc elevation compared with symptoms referred by the patients. The patients showed increased optic nerve sheath diameter [median, 6.51 mm (interquartile range 6.13-7.10)], optic nerve diameter [3.02 mm (2.86-3.27)], and optic disc elevation [0.90 mm (0.64-1.36)] at the time of admission and had ophthalmologically confirmed the presence of papilledema in all 22 patients. After 6 months all parameters decreased significantly for optic nerve sheath diameter [6.08 mm (5.59-6.73), P = 0.002], optic nerve diameter [2.87 mm (2.70-3.15), P = 0.007], and optic disc elevation [0.48 mm (0.30-0.70), P < 0.001]. In addition, a discrete negative correlation between optic nerve sheath diameter and headache change after 6 months was observed with ρ = - 0.477 and P = 0.02. No correlation was found between optic disc elevation and headache. In conclusion, longitudinal follow-up with ocular ultrasound combined with clinical information may provide support for treatment of this condition.

Impaired cholinergic transmission in patients with Parkinson's disease and olfactory dysfunction.

Olfactory dysfunction represents a frequent and disturbing non-motor manifestation of Parkinson's disease (PD). The pathophysiology of olfactory dysfunction in PD is still poorly understood. Experimental evidence suggests that olfactory impairment could be related to central cholinergic dysfunction. Short latency afferent inhibition (SAI) technique gives the opportunity to test an inhibitory cholinergic circuit in the human cerebral motor cortex. The objective of the study was to assess the cholinergic function, as measured by SAI, in PD patients with different degrees of olfactory dysfunction. We applied SAI technique in 31 patients with PD. These patients also underwent Olfactory Event-Related Potentials (OERPs) studies to objectively evaluate the olfactory system and a battery of neuropsychological tests to assess the cognitive functions. Absent OERPs indicated a severe olfactory dysfunction in 13 subjects. The presence of OERPs with an alteration in latency and/or amplitude can be considered as a borderline condition of slight alteration of smell and was found in other 15 patients. Only 3 patients showed normal OERPs. SAI was significantly reduced in the PD patients with absent OERPs compared with those with present but abnormal OERPs. Neuropsychological examination showed a mild cognitive impairment in 12 out of 13 PD patients with severe olfactory dysfunction, and in 3 out of the 15 patients with borderline olfactory dysfunction. SAI abnormalities and presence of severe olfactory impairment strongly support the hypothesis of cholinergic dysfunction in some patients with PD, who will probably develop a dementia. Longitudinal studies are required to verify whether SAI abnormalities in PD patients with olfactory dysfunction can predict a future severe cognitive decline.

Effects of intermittent theta burst stimulation on spasticity after spinal cord injury.

PURPOSE: Spasticity is a common disorder in patients with spinal cord injury (SCI). The aim of this study was to investigate whether intermittent theta burst stimulation (iTBS), a safe, non-invasive and well-tolerated protocol of excitatory repetitive transcranial magnetic stimulation (rTMS), is effective in modulating spasticity in SCI patients. METHODS: In this randomized, double-blind, crossover, sham-controlled study, ten subjects with incomplete cervical or thoracic SCI received 10 days of daily sessions of real or sham iTBS. The H/M amplitude ratio of the Soleus H reflex, the amplitude of the motor evoked potentials (MEPs) at rest and during background contraction, as well as Modified Ashworth Scale (MAS) and the Spinal Cord Injury Assessment Tool for Spasticity (SCAT) were compared before and after the stimulation protocols. RESULTS: Patients receiving real iTBS showed significant increased resting and active MEPs amplitude and a significant reduction of the H/M amplitude ratio. In these patients also the MAS and SCAT scores were significantly reduced after treatment. These changes persisted up to 1 week after the end of the iTBS treatment, and were not observed under the sham-TBS condition. CONCLUSION: These findings suggest that iTBS may be a promising therapeutic tool for the spasticity in SCI patients.

Passive cycling in neurorehabilitation after spinal cord injury: A review.

CONTEXT/OBJECTIVE: Passive cycling (PC) may represent a potential alternative neurorehabilitation program for patients who are too weak or medically unstable to repeatedly practice active movements. We review here the most important animal and human studies addressing PC after spinal cord injury (SCI). METHODS: A MEDLINE search was performed using following terms: "passive", "cycling", "pedaling", "pedalling","spinal cord injury". RESULTS: Experimental studies revealed that PC modulated spinal reflex and reduced spasticity. PC also reduced autonomic dysreflexia and elicited cardio-protective effects. Increased levels of mRNA for brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and neurotrophin-4 were found. In contrast, human studies failed to show an effect of PC on spasticity reduction and did not support its application for prevention of cardiovascular disease-related secondary complications. CONCLUSION: Available evidence to support the use of PC as standard treatment in patients with SCI is still rather limited. Since it is conceivable that PC motion could elicit sensory inputs to activate cortical structures and induce cortical plasticity changes leading to improved lower limb motor performance, further carefully designed prospective studies in subjects with SCI are needed.

Trigemino-cervical-spinal reflexes after traumatic spinal cord injury.

OBJECTIVE: After spinal cord injury (SCI) reorganization of spinal cord circuits occur both above and below the spinal lesion. These functional changes can be determined by assessing electrophysiological recording. We aimed at investigating the trigemino-cervical reflex (TCR) and trigemino-spinal reflex (TSR) responses after traumatic SCI. METHODS: TCR and TSR were registered after stimulation of the infraorbital nerve from the sternocleidomastoid, splenius, deltoid, biceps and first dorsal interosseous muscles in 10 healthy subjects and 10 subjects with incomplete cervical SCI. RESULTS: In the control subjects reflex responses were registered from the sternocleidomastoid, and splenium muscles, while no responses were obtained from upper limb muscles. In contrast, smaller but clear short latency EMG potentials were recorded from deltoid and biceps muscles in about half of the SCI patients. Moreover, the amplitudes of the EMG responses in the neck muscles were significantly higher in patients than in control subjects. CONCLUSION: The reflex responses are likely to propagate up the brainstem and down the spinal cord along the reticulospinal tracts and the propriospinal system. Despite the loss of corticospinal axons, synaptic plasticity in pre-existing pathways and/or formation of new circuits through sprouting processes above the injury site may contribute to the findings of this preliminary study and may be involved in the functional recovery. SIGNIFICANCE: Trigemino-cervical-spinal reflexes can be used to demonstrate and quantify plastic changes at brainstem and cervical level following SCI.