Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

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Abstract

Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene ( CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

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Genome-wide analysis of mammalian promoter architecture and evolution.

Piero Carninci, Albin Sandelin, Boris Lenhard … (2006)

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

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Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1

Gemma L. Carvill, Sinead B Heavin, Simone Yendle … (2013)

Introductory Epileptic encephalopathies are a devastating group of epilepsies with a poor prognosis, for which the majority have unknown etiology. We perform targeted massively parallel resequencing of 19 known and 46 candidate epileptic encephalopathy genes in 500 patients to identify novel genes and investigate the phenotypic spectrum of known genes. Overall, we identify pathogenic mutations in 10% of our cohort. Six of the 46 candidate genes had one or more pathogenic variants, collectively accounting for 3% of our cohort. We show that de novo CHD2 and SYNGAP1 mutations are novel causes of epileptic encephalopathies, accounting for 1.2% and 1% of cases respectively. We also further expand the phenotypic spectrum for SCN1A, SCN2A, and SCN8A mutations. To our knowledge, this is the largest cohort of patients with epileptic encephalopathies to undergo targeted resequencing. Implementation of this rapid and efficient method will change diagnosis and understanding of the molecular etiologies of these disorders. Epilepsy is one of the most common neurological disorders with a lifetime incidence of 3%. Epileptic encephalopathies are a devastating group of epilepsies characterized by refractory seizures and cognitive arrest or regression associated with ongoing epileptic activity, and typically carry a poor prognosis 1 . De novo mutations in several known genes are responsible for some epileptic encephalopathies 2 . Furthermore, we and others have shown that rare, de novo copy number variants (CNVs) account for up to ~8% of cases 3, 4 . Despite this recent progress, making a genetic diagnosis in a patient can be challenging as there is both genetic heterogeneity for a given epilepsy syndrome and phenotypic heterogeneity for a specific gene. The full phenotypic spectrum associated with mutations in known epileptic encephalopathy genes is not known. Very few studies have investigated the role of any given gene across a wide spectrum of epileptic encephalopathy syndromes. This makes serial gene testing in the clinical setting an inefficient and expensive process, after which the vast majority of cases remain unexplained. Furthermore, it is clear that discovery of additional genes that cause epileptic encephalopathies is needed to facilitate genetic diagnosis. Here, we take advantage of a high-throughput targeted sequencing approach to perform comprehensive sequence analysis of 65 genes (19 known genes and 46 candidate genes) (Supplementary Fig. 1) in 500 patients with a range of epileptic encephalopathy phenotypes (Table 1). Candidate genes were selected from epilepsy-associated CNVs (n=33) or because mutations cause associated neurodevelopmental disorders or other epilepsy syndromes (n=13). Using this approach, we (i) identify novel epileptic encephalopathy genes and (ii) delineate the phenotypic spectrum and mutation frequency for both known and novel epileptic encephalopathy genes. Overall, 91% of the target (65 genes) was sequenced at >25X coverage, required for accurate variant calling (Supplementary Fig. 2). We achieved 91% sensitivity across 685 variants (161 loci) from 12 samples that had previously undergone exome sequencing and 100% sensitivity for 24 known variants in previously tested patients; these patients were not included in the discovery cohort. We detected one or more pathogenic or likely pathogenic mutations in six of our 46 candidate genes, with multiple individuals carrying mutations in either of the two novel epileptic encephalopathy genes, CHD2 (NM_001271.3, NP_001262.3) and SYNGAP1 (NM_006772.2, NP_006763.2) (Table 1, 2, Fig. 1). Remarkably, we detected six de novo variants in the candidate gene, CHD2 (Fig. 1,2), selected from within the critical interval of 15q26.1 deletions detected in patients with a range of epileptic encephalopathies (Supplementary Fig. 3) 5, 6 . Four mutations lead to premature truncation of CHD2 (Table 2). Two de novo missense variants disrupt highly conserved residues within the SNF2-related helicase/ATPase domain (p.Trp548Arg and p.Leu823Pro), and are predicted to be damaging by both PolyPhen2 and SIFT. CHD2 codes for a member of the chromodomain helicase DNA-binding family of proteins and is characterized by the presence of chromatin remodeling, chromo (chromatin organization modifier) and SNF2-related helicase/ATPase domains. These domains suggest function of this protein as a chromatin remodeler 7 . While functional studies in CHD2 are limited, studies of another CHD protein family member, CHD7, have shown that the helicase domain is responsible for ATP-dependent nucleosome remodeling, an integral process in target gene regulation. Furthermore, in vivo studies of human CHD7 mutations within the helicase domain, which cause CHARGE syndrome, resulted in decreased remodeling ability 8 . These results suggest that the two de novo missense mutations described here may disrupt CHD2 function in a similar manner, while truncating mutations likely result in haploinsufficiency. The six patients with CHD2 mutations had distinctive features with a median seizure onset of 18 months (range 1–3 years, Table 2): myoclonic seizures in all, photosensitivity in three and all had ID, ranging from moderate to severe. A de novo CHD2 frameshift mutation was reported in a proband with ID and absence seizures 9 and a de novo missense mutation in an individual with autism spectrum disorder (ASD) 10 . These results suggest that mutations in CHD2 contribute to a broad spectrum of neurodevelopmental disorders. Notably, recent studies implicate de novo mutations in CHD8 in patients with ASD 11 . Interestingly, three genes of the chromodomain family (CHD2, CHD7, CHD8) have now been implicated in disorders that impact the neurodevelopmental system. Further studies of this nine-member gene family will determine the role of each across the spectrum of neurodevelopmental disorders, and provide exciting new avenues of research. We identified nine pathogenic or likely pathogenic variants in four of the 13 ‘epilepsy-associated’ genes (Fig. 1). We found five truncating variants in SYNGAP1 (Fig. 2). Patients with SYNGAP1 mutations had median seizure onset of 14 months (mean 14 months, range 6 months to 3 years) (Table 2). They had multiple seizure types, early developmental delay and subsequent regression. Outcome was poor with moderate to severe ID. SYNGAP1 mutations have been associated with ID and, although most patients have epilepsy, seizures are typically well controlled 9, 12–18 . Our study represents the first cases of epileptic encephalopathies with SYNGAP1 mutations. These observations suggest that epilepsy is a core feature of both static and progressive encephalopathies associated with SYNGAP1 mutations, and carry important implications for diagnostic testing. Variants were identified in three additional ‘epilepsy associated genes’. There were two de novo variants in MEF2C (NM_002397.4, NP_002388.2), a missense variant and a stop-loss variant (p.*464SerExt*?). Furthermore, we found de novo pathogenic variants in MBD5 (NM_018328.4, NP_060798.2) (Thr157Glnfs*4) and GABRG2 (NM_000816.3, NP_000807.2)(p.Arg323Gln) (Table 2). We detected a premature truncation mutation (p.Tyr805*) in the CNV candidate gene, HNRNPU (NM_031844.2, NP_114032.2). The p.Tyr805* change arose as a result of two consecutive single nucleotide changes c.471T>C and c.472A>T (Supplementary Fig. 4) that occur two amino acids upstream of the termination codon. Neither variant was maternally inherited; paternal DNA was not available. A recent report identified HNRNPU as a candidate for the ID and seizure phenotypes of probands with 1q44 microdeletions 19 . In addition, a de novo splice-site variant was identified in a proband with a complex neurodevelopmental phenotype including epilepsy 20 . Collectively, these data suggest that haploinsufficiency of HNRNPU is associated with epileptic encephalopathy as well as ID, though further phenotype-genotype correlation will improve our understanding of the HNRNPU phenotypic spectrum. We identified 32 variants fulfilling our criteria for pathogenicity and an additional four variants that are likely pathogenic in ten of 19 known epileptic encephalopathy genes (Fig. 1, Table 1, Table 3). We identified multiple patients with mutations in STXBP1, CDKL5, SCN1A, SCN2A, PCDH19 and KCNQ2, accounting for 69% (36/52) of all mutation-positive individuals in our cohort. We detected an additional 16 rare variants in six of these 19 known genes for which we were unable to conduct segregation analysis; it is probable that a number of these variants are also pathogenic (Supplementary Table 1). The phenotypes identified in patients with mutations in known genes are provided (Table 3), and for some we expand the known phenotypic spectrum. For example, we identified a homozygous recessive missense mutation in PNKP in a single proband with unclassified epileptic encephalopathy. PNKP mutations are associated with early infantile epileptic encephalopathy comprising microcephaly, early-onset intractable seizures and developmental delay 21 . By contrast, our patient did not have microcephaly (head circumference 50th centile) or developmental delay but had normal cognition despite refractory epilepsy with multiple seizure types. Also, three patients with SCN1A mutations presented with an epilepsy-aphasia phenotype, of which two also had FS+. SCN1A mutations are well known to be associated with genetic epilepsy with febrile seizures plus (GEFS+) but have not previously been reported with epilepsy-aphasia syndromes 22, 23 . It is possible that the SCN1A mutation is not responsible for the epilepsy-aphasia syndrome but equally it could be a modifier predisposing the individual to this group of epileptic encephalopathies. Further work is warranted to clarify this association, perhaps most effectively with exome-sequencing in these patients. We detected five variants in SCN2A, which encodes the α2 subunit of the voltage gated sodium channel. To date, the majority of SCN2A mutations have been associated with the self-limited autosomal dominant syndrome of benign familial neonatal-infantile seizures (BFNIS) 24 . Previously, only three de novo variants have been reported in patients with epileptic encephalopathies 25, 26 . Interestingly our five cases show similar variability in the range of onset seen in BFNIS with three beginning in the neonatal period (11 hours to 2 days) and two in infancy (6 weeks, 13 months). Two had relatively early offset of seizures at 5 weeks and 7 months. The refractory nature of seizures did not correlate with intellectual outcome, which ranged from mild (2) to severe (3) intellectual disability. We conclude that SCN2A is an important contributor to the overall burden of epileptic encephalopathies, accounting for 1% of cases. We also identified a pathogenic missense mutation (p.Leu1290Val) in SCN8A. To date, only a single de novo SCN8A mutation (p.Asn1768Asp) has been described in a proband with severe epileptic encephalopathy and sudden unexplained death in epilepsy 27 . Here we describe a second patient presenting with an epileptic encephalopathy beginning at 18 months. Interestingly, this variant was paternally inherited, though the father was shown to have somatic mosaicism (13% mutant allele) supporting its pathogenic effect as seen in other genetic encephalopathies with parental mosaicism 28 . The findings in this large series of patients with hitherto unsolved epileptic encephalopathies allows us to begin to frame the overall genetic architecture of this group of disorders. We identified pathogenic or likely pathogenic mutations in 10% of our cohort, with mutations in 16 genes. However, this mutation rate is likely to be an underestimation of the true contribution of each gene to the overall burden of epileptic encephalopathies. Our cohort excluded patients with previously identified mutations, and we were unable to conduct segregation analysis for a subset of variants we identified, some of which are likely to be pathogenic. Furthermore, as larger numbers of patients with mutations of specific genes are identified, distinctive epileptic encephalopathy phenotypes are likely to emerge. Taken together, with up to 8% rare CNVs in epileptic encephalopathy patients in an earlier analysis of a subset of this series 3 , we can now collectively ascribe causality for ~18% of all epileptic encephalopathies of unknown cause. The genetic heterogeneity of epileptic encephalopathies is considerable; likely pathogenic variants were found in nine known or novel genes (see Fig. 2). Even the most commonly mutated genes in our study each account for only up to 1.6% of cases. Notably, we elucidate new genes found to be commonly mutated in epileptic encephalopathies, with CHD2, SYNGAP1 and SCN2A accounting for 1–1.2% of cases each, a frequency similar to that of mutations in SCN1A, STXBP1 and CDKL5 in our cohort. However, no mutations were seen in nine other known genes (ARX, FOXG1, KCNT1, MECP2, PLCB1, SLC25A22, SLC2A1, SPTAN1, ARHGEF9) in 500 patients. These results suggest that pathogenic mutations in these genes, while important, are rare causes of epileptic encephalopathies ( 0.70, QUAL 25X for each gene. Rare variant segregation analysis Where family members were available, segregation analysis was carried out for all rare (not present in ESP6500 controls), possibly damaging (non-synonymous, essential splice-site or frameshift) variants for all 65 target genes. This analysis was performed using a ‘MIP-pick’ strategy. We selected and re-pooled only the MIPs that captured the genomic sequence harboring the rare variant of interest and performed target enrichment PCR and sequencing as above for all relevant probands and family members. This approach allowed us to sequence variants at very high depth and detect somatic mosaicism in parents. Criteria for pathogenicity of rare variants For those rare, possibly damaging variants where segregation analysis could be performed, we required the variant to meet one of the following criteria to constitute a novel pathogenic variant. Pathogenic variants: (i) arose de novo, (ii) segregated with the disorder, (iii) were inherited from a parent with somatic mosaicism, or (iv) adhered to a recessive, X-linked or parent-of-origin mode of inheritance, where applicable (Supplementary Fig. 1). In certain instances we were unable to determine the inheritance of a rare variant due to the unavailability of DNA from one or more parent. It is likely that a subset of these variants also cause disease, though here we report only those variants that are likely to lead to protein truncation (i.e. splice-site, nonsense, frameshift, stop-loss) as being ‘likely pathogenic’. Additionally, two missense mutations in known genes (STXBP1, SCN2A) were interpreted to be ‘likely pathogenic’ based on the high incidence of pathogenic missense mutations in these genes, which was further supported by the available parent not carrying the variant. We performed microsatellite analysis using the PowerPlex S5 system [Promega] in all parents of probands with a de novo mutation to confirm maternity and paternity. Supplementary Material 1

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Comparison of the transcriptional landscapes between human and mouse tissues.

Shin Lin, Yiing Lin, Joseph Nery … (2014)

Although the similarities between humans and mice are typically highlighted, morphologically and genetically, there are many differences. To better understand these two species on a molecular level, we performed a comparison of the expression profiles of 15 tissues by deep RNA sequencing and examined the similarities and differences in the transcriptome for both protein-coding and -noncoding transcripts. Although commonalities are evident in the expression of tissue-specific genes between the two species, the expression for many sets of genes was found to be more similar in different tissues within the same species than between species. These findings were further corroborated by associated epigenetic histone mark analyses. We also find that many noncoding transcripts are expressed at a low level and are not detectable at appreciable levels across individuals. Moreover, the majority lack obvious sequence homologs between species, even when we restrict our attention to those which are most highly reproducible across biological replicates. Overall, our results indicate that there is considerable RNA expression diversity between humans and mice, well beyond what was described previously, likely reflecting the fundamental physiological differences between these two organisms.

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Ruben Artero: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 17 June 2016

Publication date Collection: 2016

Volume: 11

Issue: 6

Electronic Location Identifier: e0157758

Affiliations

[1 ]Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom

[2 ]Patrick Wild Centre, University of Edinburgh, Edinburgh, United Kingdom

[3 ]Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom

[4 ]Centre for Brain Development and Repair, The Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, India

[5 ]Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy

[6 ]Department of Theoretical and Applied Sciences, Division of Biomedical Research, University of Insubria, Busto Arsizio, Italy

[7 ]School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom

University of Valencia, SPAIN

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: RDH OD NL CKN PCK MESB SRC. Performed the experiments: RDH OD. Analyzed the data: RDH OD. Contributed reagents/materials/analysis tools: RDH OD NL CKN. Wrote the paper: RDH OD NL CKN PCK MESB SRC.

* E-mail: stuart.cobb@ 123456glasgow.ac.uk

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Ralph D. Hector http://orcid.org/0000-0002-3778-1483

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Publisher ID: PONE-D-16-05131

DOI: 10.1371/journal.pone.0157758

PMC ID: 4912119

PubMed ID: 27315173

SO-VID: c2d2f3a9-e1eb-439c-b2b0-11319ab2896f

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This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 8 February 2016

Date accepted : 3 June 2016

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Figures: 9, Tables: 3, Pages: 22

Funding

Funded by: funder-id http://dx.doi.org/10.13039/501100000589, Chief Scientist Office;

Award ID: ETM/334

Award Recipient : Stuart R. Cobb

Funded by: funder-id http://dx.doi.org/10.13039/501100000589, Chief Scientist Office;

Award ID: ETM/334

Award Recipient : Mark E. S. Bailey

The Chief Scientist Office of the Scottish Government (grant ETM/334) and the Rett Syndrome Association Scotland provided funding to SRC and MESB. NL and CKN are supported by the Italian parents’ association L’Albero di Greta. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

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Most cited references 38

Genome-wide analysis of mammalian promoter architecture and evolution.

Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1

Comparison of the transcriptional landscapes between human and mouse tissues.

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