Chronic intestinal pseudo-obstruction in adults: A practical guide to identify patient subgroups that are suitable for more specific treatments.

Chronic intestinal pseudo-obstruction is a rare and heterogeneous syndrome characterized by recurrent symptoms of intestinal obstruction with radiological features of dilated small or large intestine with air/fluid levels in the absence of any mechanical occlusive lesion. Several diseases may be associated with chronic intestinal pseudo-obstruction and in these cases, the prognosis and treatment are related to the underlying disease. Also, in its "primary or idiopathic" form, two subgroups of patients should be determined as they require a more specific therapeutic approach: patients whose chronic intestinal pseudo-obstruction is due to sporadic autoimmune/inflammatory mechanisms and patients whose neuromuscular changes are genetically determined. In a context of a widely heterogeneous adult population presenting chronic intestinal pseudo-obstruction, this review aims to summarize a practical diagnostic workup for identifying definite subgroups of patients who might benefit from more specific treatments, based on the etiology of their underlying condition.

Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations.

Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.

Genetic Profiling of Sodium Channels in Diabetic Painful and Painless and Idiopathic Painful and Painless Neuropathies.

Neuropathic pain is a frequent feature of diabetic peripheral neuropathy (DPN) and small fiber neuropathy (SFN). Resolving the genetic architecture of these painful neuropathies will lead to better disease management strategies, counselling and intervention. Our aims were to profile ten sodium channel genes (SCG) expressed in a nociceptive pathway in painful and painless DPN and painful and painless SFN patients, and to provide a perspective for clinicians who assess patients with painful peripheral neuropathy. Between June 2014 and September 2016, 1125 patients with painful-DPN (n = 237), painless-DPN (n = 309), painful-SFN (n = 547) and painless-SFN (n = 32), recruited in four different centers, were analyzed for SCN3A, SCN7A-SCN11A and SCN1B-SCN4B variants by single molecule Molecular inversion probes-Next Generation Sequence. Patients were grouped based on phenotype and the presence of SCG variants. Screening of SCN3A, SCN7A-SCN11A, and SCN1B-SCN4B revealed 125 different (potential) pathogenic variants in 194 patients (17.2%, n = 194/1125). A potential pathogenic variant was present in 18.1% (n = 142/784) of painful neuropathy patients vs. 15.2% (n = 52/341) of painless neuropathy patients (17.3% (n = 41/237) for painful-DPN patients, 14.9% (n = 46/309) for painless-DPN patients, 18.5% (n = 101/547) for painful-SFN patients, and 18.8% (n = 6/32) for painless-SFN patients). Of the variants detected, 70% were in SCN7A, SCN9A, SCN10A and SCN11A. The frequency of SCN9A and SCN11A variants was the highest in painful-SFN patients, SCN7A variants in painful-DPN patients, and SCN10A variants in painless-DPN patients. Our findings suggest that rare SCG genetic variants may contribute to the development of painful neuropathy. Genetic profiling and SCG variant identification should aid in a better understanding of the genetic variability in patients with painful and painless neuropathy, and may lead to better risk stratification and the development of more targeted and personalized pain treatments.

Integrative miRNA-mRNA profiling of human epidermis: unique signature of SCN9A painful neuropathy.

Personalized management of neuropathic pain is an unmet clinical need due to heterogeneity of the underlying aetiologies, incompletely understood pathophysiological mechanisms and limited efficacy of existing treatments. Recent studies on microRNA in pain preclinical models have begun to yield insights into pain-related mechanisms, identifying nociception-related species differences and pinpointing potential drug candidates. With the aim of bridging the translational gap towards the clinic, we generated a human pain-related integrative miRNA and mRNA molecular profile of the epidermis, the tissue hosting small nerve fibres, in a deeply phenotyped cohort of patients with sodium channel-related painful neuropathy not responding to currently available therapies. We identified four miRNAs strongly discriminating patients from healthy individuals, confirming their effect on differentially expressed gene targets driving peripheral sensory transduction, transmission, modulation and post-transcriptional modifications, with strong effects on gene targets including NEDD4. We identified a complex epidermal miRNA-mRNA network based on tissue-specific experimental data suggesting a cross-talk between epidermal cells and axons in neuropathy pain. Using immunofluorescence assay and confocal microscopy, we observed that Nav1.7 signal intensity in keratinocytes strongly inversely correlated with NEDD4 expression that was downregulated by miR-30 family, suggesting post-transcriptional fine tuning of pain-related protein expression. Our targeted molecular profiling advances the understanding of specific neuropathic pain fine signatures and may accelerate process towards personalized medicine in patients with neuropathic pain.

Peripheral Ion Channel Genes Screening in Painful Small Fiber Neuropathy.

Neuropathic pain is a characteristic feature of small fiber neuropathy (SFN), which in 18% of the cases is caused by genetic variants in voltage-gated sodium ion channels. In this study, we assessed the role of fifteen other ion channels in neuropathic pain. Patients with SFN (n = 414) were analyzed for ANO1, ANO3, HCN1, KCNA2, KCNA4, KCNK18, KCNN1, KCNQ3, KCNQ5, KCNS1, TRPA1, TRPM8, TRPV1, TRPV3 and TRPV4 variants by single-molecule molecular inversion probes-next-generation sequencing. These patients did not have genetic variants in SCN3A, SCN7A-SCN11A and SCN1B-SCN4B. In twenty patients (20/414, 4.8%), a potentially pathogenic heterozygous variant was identified in an ion-channel gene (ICG). Variants were present in seven genes, for two patients (0.5%) in ANO3, one (0.2%) in KCNK18, two (0.5%) in KCNQ3, seven (1.7%) in TRPA1, three (0.7%) in TRPM8, three (0.7%) in TRPV1 and two (0.5%) in TRPV3. Variants in the TRP genes were the most frequent (n = 15, 3.6%), partly in patients with high mean maximal pain scores VAS = 9.65 ± 0.7 (n = 4). Patients with ICG variants reported more severe pain compared to patients without such variants (VAS = 9.36 ± 0.72 vs. VAS = 7.47 ± 2.37). This cohort study identified ICG variants in neuropathic pain in SFN, complementing previous findings of ICG variants in diabetic neuropathy. These data show that ICG variants are central in neuropathic pain of different etiologies and provides promising gene candidates for future research.

Peripheral Ion Channel Gene Screening in Painful- and Painless-Diabetic Neuropathy.

Neuropathic pain is common in diabetic peripheral neuropathy (DN), probably caused by pathogenic ion channel gene variants. Therefore, we performed molecular inversion probes-next generation sequencing of 5 transient receptor potential cation channels, 8 potassium channels and 2 calcium-activated chloride channel genes in 222 painful- and 304 painless-DN patients. Twelve painful-DN (5.4%) patients showed potentially pathogenic variants (five nonsense/frameshift, seven missense, one out-of-frame deletion) in ANO3 (n = 3), HCN1 (n = 1), KCNK18 (n = 2), TRPA1 (n = 3), TRPM8 (n = 3) and TRPV4 (n = 1) and fourteen painless-DN patients (4.6%-three nonsense/frameshift, nine missense, one out-of-frame deletion) in ANO1 (n = 1), KCNK18 (n = 3), KCNQ3 (n = 1), TRPA1 (n = 2), TRPM8 (n = 1), TRPV1 (n = 3) and TRPV4 (n = 3). Missense variants were present in both conditions, presumably with loss- or gain-of-functions. KCNK18 nonsense/frameshift variants were found in painless/painful-DN, making a causal role in pain less likely. Surprisingly, premature stop-codons with likely nonsense-mediated RNA-decay were more frequent in painful-DN. Although limited in number, painful-DN patients with ion channel gene variants reported higher maximal pain during the night and day. Moreover, painful-DN patients with TRP variants had abnormal thermal thresholds and more severe pain during the night and day. Our results suggest a role of ion channel gene variants in neuropathic pain, but functional validation is required.

Congenital insensitivity to pain: a novel mutation affecting a U12-type intron causes multiple aberrant splicing of SCN9A.

ABSTRACT: Mutations in the alpha subunit of voltage-gated sodium channel 1.7 (NaV1.7), encoded by SCN9A gene, play an important role in the regulation of nociception and can lead to a wide range of clinical outcomes, ranging from extreme pain syndromes to congenital inability to experience pain. To expand the phenotypic and genotypic spectrum of SCN9A-related channelopathies, we describe the proband, a daughter born from consanguineous parents, who had pain insensitivity, diminished temperature sensation, foot burns, and severe loss of nociceptive nerve fibers in the epidermis. Next-generation sequencing of SCN9A (NM_002977.3) revealed a novel homozygous substitution (c.377+7T>G) in the donor splice site of intron 3. As the RNA functional testing is challenging, the in silico analysis is the first approach to predict possible alterations. In this case, the computational analysis was unable to identify the splicing consensus and could not provide any prediction for splicing defects. The affected intron indeed belongs to the U12 type, a family of introns characterised by noncanonical consensus at the splice sites, accounting only for 0.35% of all human introns, and is not included in most of the training sets for splicing prediction. A functional study on proband RNA showed different aberrant transcripts, where exon 3 was missing and an intron fragment was included. A quantification study using real-time polymerase chain reaction showed a significant reduction of the NaV1.7 canonical transcript. Collectively, these data widen the spectrum of SCN9A-related insensitivity to pain by describing a mutation causing NaV1.7 deficiency, underlying the nociceptor dysfunction, and highlight the importance of molecular investigation of U12 introns' mutations despite the silent prediction.

Correction: Evaluation of molecular inversion probe versus TruSeq® custom methods for targeted next-generation sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0238467.].

Computational pipeline to probe NaV1.7 gain-of-function variants in neuropathic painful syndromes.

Applications of machine learning and graph theory techniques to neuroscience have witnessed an increased interest in the last decade due to the large data availability and unprecedented technology developments. Their employment to investigate the effect of mutational changes in genes encoding for proteins modulating the membrane of excitable cells, whose biological correlates are assessed at electrophysiological level, could provide useful predictive clues. We apply this concept to the analysis of variants in sodium channel NaV1.7 subunit found in patients with chronic painful syndromes, by the implementation of a dedicated computational pipeline empowering different and complementary techniques including homology modeling, network theory, and machine learning. By testing three templates of different origin and sequence identities, we provide an optimal condition for its use. Our findings reveal the usefulness of our computational pipeline in supporting the selection of candidates for cell electrophysiology assay and with potential clinical applications.

Evaluation of molecular inversion probe versus TruSeq® custom methods for targeted next-generation sequencing.

Resolving the genetic architecture of painful neuropathy will lead to better disease management strategies. We aimed to develop a reliable method to re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. In this study, we compared sensitivity, specificity, targeting efficiency, performance and cost effectiveness of Molecular Inversion Probes-Next generation sequencing (MIPs-NGS) and TruSeq® Custom Amplicon-Next generation sequencing (TSCA-NGS). Capture probes were designed to target nine sodium channel genes (SCN3A, SCN8A-SCN11A, and SCN1B-SCN4B). One hundred sixty-six patients with diabetic and idiopathic neuropathy were tested by both methods, 70 patients were validated by Sanger sequencing. Sensitivity, specificity and performance of both techniques were comparable, and in agreement with Sanger sequencing. The average targeted regions coverage for MIPs-NGS was 97.3% versus 93.9% for TSCA-NGS. MIPs-NGS has a more versatile assay design and is more flexible than TSCA-NGS. The cost of MIPs-NGS is >5 times cheaper than TSCA-NGS when 500 or more samples are tested. In conclusion, MIPs-NGS is a reliable, flexible, and relatively inexpensive method to detect genetic variations in a large cohort of patients. In our centers, MIPs-NGS is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients.

Diagnostic criteria for small fibre neuropathy in clinical practice and research.

The diagnostic criteria for small fibre neuropathy are not established, influencing the approach to patients in clinical practice, their access to disease-modifying and symptomatic treatments, the use of healthcare resources, and the design of clinical trials. To address these issues, we performed a reappraisal study of 150 patients with sensory neuropathy and a prospective and follow-up validation study of 352 new subjects with suspected sensory neuropathy. Small fibre neuropathy diagnostic criteria were based on deep clinical phenotyping, quantitative sensory testing (QST) and intraepidermal nerve fibre density (IENFD). Small fibre neuropathy was ruled out in 5 of 150 patients (3.3%) of the reappraisal study. Small fibre neuropathy was diagnosed at baseline of the validation study in 149 of 352 patients (42.4%) based on the combination between two clinical signs and abnormal QST and IENFD (69.1%), abnormal QST alone (5.4%), or abnormal IENFD alone (20.1%). Eight patients (5.4%) had abnormal QST and IENFD but no clinical signs. Further, 38 patients complained of sensory symptoms but showed no clinical signs. Of those, 34 (89.4%) had normal QST and IENFD, 4 (10.5%) had abnormal QST and normal IENFD, and none had abnormal IENFD alone. At 18-month follow-up, 19 of them (56%) reported the complete recovery of symptoms and showed normal clinical, QST and IENFD findings. None of those with one single abnormal test (QST or IENFD) developed clinical signs or showed abnormal findings on the other test. Conversely, all eight patients with abnormal QST and IENFD at baseline developed clinical signs at follow-up. The combination of clinical signs and abnormal QST and/or IENFD findings can more reliably lead to the diagnosis of small fibre neuropathy than the combination of abnormal QST and IENFD findings in the absence of clinical signs. Sensory symptoms alone should not be considered a reliable screening feature. Our findings demonstrate that the combined clinical, functional and structural approach to the diagnosis of small fibre neuropathy is reliable and relevant both for clinical practice and clinical trial design.

Pathogenic Variants in STXBP1 and in Genes for GABAa Receptor Subunities Cause Atypical Rett/Rett-like Phenotypes.

Rett syndrome (RTT) is a neurodevelopmental disorder, affecting 1 in 10,000 girls. Intellectual disability, loss of speech and hand skills with stereotypies, seizures and ataxia are recurrent features. Stringent diagnostic criteria distinguish classical Rett, caused by a MECP2 pathogenic variant in 95% of cases, from atypical girls, 40-73% carrying MECP2 variants, and rarely CDKL5 and FOXG1 alterations. A large fraction of atypical and RTT-like patients remain without genetic cause. Next Generation Sequencing (NGS) targeted to multigene panels/Whole Exome Sequencing (WES) in 137 girls suspected for RTT led to the identification of a de novo variant in STXBP1 gene in four atypical RTT and two RTT-like girls. De novo pathogenic variants-one in GABRB2 and, for first time, one in GABRG2-were disclosed in classic and atypical RTT patients. Interestingly, the GABRG2 variant occurred at low rate percentage in blood and buccal swabs, reinforcing the relevance of mosaicism in neurological disorders. We confirm the role of STXBP1 in atypical RTT/RTT-like patients if early psychomotor delay and epilepsy before 2 years of age are observed, indicating its inclusion in the RTT diagnostic panel. Lastly, we report pathogenic variants in Gamma-aminobutyric acid-A (GABAa) receptors as a cause of atypical/classic RTT phenotype, in accordance with the deregulation of GABAergic pathway observed in MECP2 defective in vitro and in vivo models.

Yield of peripheral sodium channels gene screening in pure small fibre neuropathy.

BACKGROUND: Neuropathic pain is common in peripheral neuropathy. Recent genetic studies have linked pathogenic voltage-gated sodium channel (VGSC) variants to human pain disorders. Our aims are to determine the frequency of SCN9A, SCN10A and SCN11A variants in patients with pure small fibre neuropathy (SFN), analyse their clinical features and provide a rationale for genetic screening. METHODS: Between September 2009 and January 2017, 1139 patients diagnosed with pure SFN at our reference centre were screened for SCN9A, SCN10A and SCN11A variants. Pathogenicity of variants was classified according to established guidelines of the Association for Clinical Genetic Science and frequencies were determined. Patients with SFN were grouped according to the VGSC variants detected, and clinical features were compared. RESULTS: Among 1139 patients with SFN, 132 (11.6%) patients harboured 73 different (potentially) pathogenic VGSC variants, of which 50 were novel and 22 were found in ≥ 1 patient. The frequency of (potentially) pathogenic variants was 5.1% (n=58/1139) for SCN9A, 3.7% (n=42/1139) for SCN10A and 2.9% (n=33/1139) for SCN11A. Only erythromelalgia-like symptoms and warmth-induced pain were significantly more common in patients harbouring VGSC variants. CONCLUSION: (Potentially) pathogenic VGSC variants are present in 11.6% of patients with pure SFN. Therefore, genetic screening of SCN9A, SCN10A and SCN11A should be considered in patients with pure SFN, independently of clinical features or underlying conditions.

A novel SCN9A splicing mutation in a compound heterozygous girl with congenital insensitivity to pain, hyposmia and hypogeusia.

Congenital insensitivity to pain (CIP) is a rare autosomal recessive disorder presenting with a spectrum of clinical features caused by mutations in different genes. A 10-year-old girl with CIP, hyposmia and hypogeusia, and her unaffected twin and parents underwent next generation sequencing of SCN9A exons and flanking splice sites. Transcript analysis from whole blood successfully assayed the effect of the mutation on the mRNA splicing by polymerase chain reaction amplification on cDNA and Sanger sequencing. We identified the novel splicing variant c.1108-2A>G compound with the p.Arg896Gln (c.2687G>A) missense mutation previously described in a homozygous patient. The new intronic variant was predicted to induce exon 10 skipping. Conversely, SCN9A mRNA assay demonstrated its partial deletion with a loss of 46 nucleotides causing a premature stop codon in position p.Gln369 (NP_002968). Genetic analysis showed that the two variants were biallelic, being the mother and brother heterozygous carriers of the missense mutation, and the father heterozygous for the splicing mutation. Skin biopsy showed lack of Meissner's corpuscles, loss of epidermal nociceptors and normal autonomic organ innervation. We report a novel splicing mutation and provide clues on its pathogenic effect, broadening the spectrum of genotypes and phenotypes associated to CIP.

Laser capture microdissection for transcriptomic profiles in human skin biopsies.

BACKGROUND: The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data. RESULTS: The protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater® Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number (RIN). RNA was then enriched using the TruSeq® RNA Access Library Prep Kit (Illumina®) and sequenced on HiSeq® 2500 platform (Illumina®). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis. CONCLUSIONS: The described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.

COL6A5 variants in familial neuropathic chronic itch.

Itch is thought to represent the peculiar response to stimuli conveyed by somatosensory pathways shared with pain through the activation of specific neurons and receptors. It can occur in association with dermatological, systemic and neurological diseases, or be the side effect of certain drugs. However, some patients suffer from chronic idiopathic itch that is frequently ascribed to psychological distress and for which no biomarker is available to date. We investigated three multigenerational families, one of which diagnosed with joint hypermobility syndrome/Ehlers-Danlos syndrome hypermobility type (JHS/EDS-HT), characterized by idiopathic chronic itch with predominantly proximal distribution. Skin biopsy was performed in all eight affected members and revealed in six of them reduced intraepidermal nerve fibre density consistent with small fibre neuropathy. Whole exome sequencing identified two COL6A5 rare variants co-segregating with chronic itch in eight affected members and absent in non-affected members, and in one unrelated sporadic patient with type 1 painless diabetic neuropathy and chronic itch. Two families and the diabetic patient carried the nonsense c.6814G>T (p.Glu2272*) variant and another family carried the missense c.6486G>C (p.Arg2162Ser) variant. Both variants were predicted as likely pathogenic by in silico analyses. The two variants were rare (minor allele frequency < 0.1%) in 6271 healthy controls and absent in 77 small fibre neuropathy and 167 JHS/EDS-HT patients without itch. Null-allele test on cDNA from patients' fibroblasts of both families carrying the nonsense variant demonstrated functional haploinsufficiency due to activation of nonsense mediated RNA decay. Immunofluorescence microscopy and western blotting revealed marked disorganization and reduced COL6A5 synthesis, respectively. Indirect immunofluorescence showed reduced COL6A5 expression in the skin of patients carrying the nonsense variant. Treatment with gabapentinoids provided satisfactory itch relief in the patients carrying the mutations. Our findings first revealed an association between COL6A5 gene and familiar chronic itch, suggesting a new contributor to the pathogenesis of neuropathic itch and identifying a new candidate therapeutic target.

Role of UBE3A and ATP10A genes in autism susceptibility region 15q11-q13 in an Italian population: a positive replication for UBE3A.

The aetiology of autism is still largely unknown despite analyses from family and twin studies demonstrating substantial genetic role in the aetiology of the disorder. Data from linkage studies and analyses of chromosomal abnormalities identified 15q11-q13 as a region of particular aetiopathogenesis interest. We screened a set of markers spanning two known imprinted, maternally expressed genes, UBE3A and ATP10A, harboured in this candidate region. We replicated evidence of linkage disequilibrium (LD) at marker D15S122, located at the 5' end of UBE3A and originally reported by Nurmi et al. (2001). The potential role of UBE3A in our family-based association study is further supported by the association of two haplotypes that include one of the alleles of D15S122 and by the transmission disequilibrium test (TDT) evidence of the same allele in a parent of origin effect analysis. In a secondary analysis, we provided the first evidence of a significant association between first word delay and psychomotor regression with the 15q11-q13 region. Our data support a potential role of UBE3A in the complex pathogenic mechanisms of autism.

Genetic investigations on 8 patients affected by ring 20 chromosome syndrome.

BACKGROUND: Mosaic Chromosome 20 ring [r(20)] is a chromosomal disorder associated with a rare syndrome characterized by a typical seizure phenotype, a particular electroclinical pattern, cognitive impairment, behavioural problems and absence of a consistent pattern of dysmorphology. The pathogenic mechanism underlying seizures disorders in r(20) syndrome is still unknown. We performed a detailed clinical and genetic study on 8 patients with r(20) chromosome, aimed at detecting the genetic mechanism underlying r(20) syndrome. METHODS: We submitted 8 subjects with a previous diagnosis of ring 20 chromosome mosaicism to a clinical re-evaluation, followed by cytogenetic, FISH, array-CGH and molecular analyses. The genetic study was also extended to their available parents. RESULTS: FISH and array-CGH experiments indicate that cryptic deletions on chromosome 20 are not the cause of the r(20) chromosome associated disease. Moreover, no evidence of chromosome 20 uniparental disomy was found. Analysis of FISH signals given by variant in size alphoid tandem repeats probes on the normal chromosome 20 and the r(20) chromosome in the mosaic carriers suggests that the r(20) chromosome is the same chromosome not circularized in the "normal" cell line. CONCLUSIONS: Higher percentages of r(20) chromosome cells were observed to be related with precocious age at seizure onset and with resistance to antiepileptic drug treatment. Behavioural problems also seem to be associated with higher percentages of r(20) chromosome cells. Our results suggest that an epigenetic mechanism perturbing the expression of genes close to the telomeric regions, rather than deletion of genes located at the distal 20p and/or 20q regions, may underlie the manifestation of r(20) syndrome.