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1.  The First Myriapod Genome Sequence Reveals Conservative Arthropod Gene Content and Genome Organisation in the Centipede Strigamia maritima 
Chipman, Ariel D. | Ferrier, David E. K. | Brena, Carlo | Qu, Jiaxin | Hughes, Daniel S. T. | Schröder, Reinhard | Torres-Oliva, Montserrat | Znassi, Nadia | Jiang, Huaiyang | Almeida, Francisca C. | Alonso, Claudio R. | Apostolou, Zivkos | Aqrawi, Peshtewani | Arthur, Wallace | Barna, Jennifer C. J. | Blankenburg, Kerstin P. | Brites, Daniela | Capella-Gutiérrez, Salvador | Coyle, Marcus | Dearden, Peter K. | Du Pasquier, Louis | Duncan, Elizabeth J. | Ebert, Dieter | Eibner, Cornelius | Erikson, Galina | Evans, Peter D. | Extavour, Cassandra G. | Francisco, Liezl | Gabaldón, Toni | Gillis, William J. | Goodwin-Horn, Elizabeth A. | Green, Jack E. | Griffiths-Jones, Sam | Grimmelikhuijzen, Cornelis J. P. | Gubbala, Sai | Guigó, Roderic | Han, Yi | Hauser, Frank | Havlak, Paul | Hayden, Luke | Helbing, Sophie | Holder, Michael | Hui, Jerome H. L. | Hunn, Julia P. | Hunnekuhl, Vera S. | Jackson, LaRonda | Javaid, Mehwish | Jhangiani, Shalini N. | Jiggins, Francis M. | Jones, Tamsin E. | Kaiser, Tobias S. | Kalra, Divya | Kenny, Nathan J. | Korchina, Viktoriya | Kovar, Christie L. | Kraus, F. Bernhard | Lapraz, François | Lee, Sandra L. | Lv, Jie | Mandapat, Christigale | Manning, Gerard | Mariotti, Marco | Mata, Robert | Mathew, Tittu | Neumann, Tobias | Newsham, Irene | Ngo, Dinh N. | Ninova, Maria | Okwuonu, Geoffrey | Ongeri, Fiona | Palmer, William J. | Patil, Shobha | Patraquim, Pedro | Pham, Christopher | Pu, Ling-Ling | Putman, Nicholas H. | Rabouille, Catherine | Ramos, Olivia Mendivil | Rhodes, Adelaide C. | Robertson, Helen E. | Robertson, Hugh M. | Ronshaugen, Matthew | Rozas, Julio | Saada, Nehad | Sánchez-Gracia, Alejandro | Scherer, Steven E. | Schurko, Andrew M. | Siggens, Kenneth W. | Simmons, DeNard | Stief, Anna | Stolle, Eckart | Telford, Maximilian J. | Tessmar-Raible, Kristin | Thornton, Rebecca | van der Zee, Maurijn | von Haeseler, Arndt | Williams, James M. | Willis, Judith H. | Wu, Yuanqing | Zou, Xiaoyan | Lawson, Daniel | Muzny, Donna M. | Worley, Kim C. | Gibbs, Richard A. | Akam, Michael | Richards, Stephen
PLoS Biology  2014;12(11):e1002005.
Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.
Author Summary
Arthropods are the most abundant animals on earth. Among them, insects clearly dominate on land, whereas crustaceans hold the title for the most diverse invertebrates in the oceans. Much is known about the biology of these groups, not least because of genomic studies of the fruit fly Drosophila, the water flea Daphnia, and other species used in research. Here we report the first genome sequence from a species belonging to a lineage that has previously received very little attention—the myriapods. Myriapods were among the first arthropods to invade the land over 400 million years ago, and survive today as the herbivorous millipedes and venomous centipedes, one of which—Strigamia maritima—we have sequenced here. We find that the genome of this centipede retains more characteristics of the presumed arthropod ancestor than other sequenced insect genomes. The genome provides access to many aspects of myriapod biology that have not been studied before, suggesting, for example, that they have diversified receptors for smell that are quite different from those used by insects. In addition, it shows specific consequences of the largely subterranean life of this particular species, which seems to have lost the genes for all known light-sensing molecules, even though it still avoids light.
PMCID: PMC4244043  PMID: 25423365
2.  Genetic Alterations Associated With Progression From Pancreatic Intraepithelial Neoplasia to Invasive Pancreatic Tumor 
Gastroenterology  2013;145(5):1098-1109.e1.
Background & Aims
Increasing grade of pancreatic intraepithelial neoplasia (PanIN) has been associated with progression to pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that control progression from PanINs to PDAC are not well understood. We investigated the genetic alterations involved in this process.
Genomic DNA samples from laser-capture microdissected PDACs and adjacent PanIN2 and PanIN3 lesions from 10 patients with pancreatic cancer were analyzed by exome sequencing.
Similar numbers of somatic mutations were identified in PanINs and tumors, but the mutational load varied greatly among cases. Ten of the 15 isolated PanINs shared more than 50% of somatic mutations with associated tumors. Mutations common to tumors and clonally related PanIN2 and PanIN3 lesions were identified as genes that could promote carcinogenesis. KRAS and TP53 were frequently altered in PanINs and tumors, but few other recurrently modified genes were detected. Mutations in DNA damage response genes were prevalent in all samples. Genes that encode proteins involved in gap junctions, the actin cytoskeleton, the mitogen-activated protein kinase signaling pathway, axon guidance, and cell cycle regulation were among the earliest targets of mutagenesis in PanINs that progressed to PDAC.
Early-stage PanIN2 lesions appear to contain many of the somatic gene alterations required for PDAC development.
PMCID: PMC3926442  PMID: 23912084
pancreas; tumorigenesis; LCM; whole genome amplification
3.  Exome sequencing identification of a GJB1 missense mutation in a kindred with X-linked spinocerebellar ataxia (SCA-X1) 
Human Molecular Genetics  2013;22(21):4329-4338.
We undertook a gene identification and molecular characterization project in a large kindred originally clinically diagnosed with SCA-X1. While presenting with ataxia, this kindred also had some unique peripheral nervous system features. The implicated region on the X chromosome was delineated using haplotyping. Large deletions and duplications were excluded by array comparative genomic hybridization. Exome sequencing was undertaken in two affected subjects. The single identified X chromosome candidate variant was then confirmed to co-segregate appropriately in all affected, carrier and unaffected family members by Sanger sequencing. The variant was confirmed to be novel by comparison with dbSNP, and filtering for a minor allele frequency of <1% in 1000 Genomes project, and was not present in the NHLBI Exome Sequencing Project or a local database at the BCM HGSC. Functional experiments on transfected cells were subsequently undertaken to assess the biological effect of the variant in vitro. The variant identified consisted of a previously unidentified non-synonymous variant, GJB1 p.P58S, in the Connexin 32/Gap Junction Beta 1 gene. Segregation studies with Sanger sequencing confirmed the presence of the variant in all affected individuals and one known carrier, and the absence of the variant in unaffected members. Functional studies confirmed that the p.P58S variant reduced the number and size of gap junction plaques, but the conductance of the gap junctions was unaffected. Two X-linked ataxias have been associated with genetic loci, with the first of these recently characterized at the molecular level. This represents the second kindred with molecular characterization of X-linked ataxia, and is the first instance of a previously unreported GJB1 mutation with a dominant and permanent ataxia phenotype, although different CNS deficits have previously been reported. This pedigree has also been relatively unique in its phenotype due to the presence of central and peripheral neural abnormalities. Other X-linked SCAs with unique features might therefore also potentially represent variable phenotypic expression of other known neurological entities.
PMCID: PMC3792691  PMID: 23773993
4.  Acquired Neuromyotonia Heralding Recurrent Thymoma in Myasthenia Gravis 
JAMA neurology  2013;70(10):1311-1314.
Acquired neuromyotonia is increasingly recognized as an autoimmune disorder, frequently associated with antibodies against voltage-gated potassium channel complex proteins. We present a case of acquired neuromyotonia as the heralding symptom of recurrent thymoma in a patient with myasthenia gravis.
A report of a single case of a 53-year-old man with myasthenia gravis and a prior thymectomy presenting with 2 months of diffuse, involuntary muscle twitching in the absence of myasthenic symptoms, electrophysiologically confirmed to be neuromyotonia. Further evaluation revealed the recurrence of malignant thymoma, accompanied by refractory arrhythmia. Serologic and cerebrospinal fluid testing confirmed the presence of antibodies directed against 2 voltage-gated potassium channel–associated proteins: LGI1 and Caspr2.
This case highlights the overlap of myasthenia, neuromyotonia, and thymoma, emphasizing the importance of appropriate tumor screening in the presence of either of the former 2 conditions.
PMCID: PMC4096576  PMID: 23978943
5.  Modality-Based Organization of Ascending Somatosensory Axons in the Direct Dorsal Column Pathway 
The Journal of Neuroscience  2013;33(45):17691-17709.
The long-standing doctrine regarding the functional organization of the direct dorsal column (DDC) pathway is the “somatotopic map” model, which suggests that somatosensory afferents are primarily organized by receptive field instead of modality. Using modality-specific genetic tracing, here we show that ascending mechanosensory and proprioceptive axons, two main types of the DDC afferents, are largely segregated into a medial–lateral pattern in the mouse dorsal column and medulla. In addition, we found that this modality-based organization is likely to be conserved in other mammalian species, including human. Furthermore, we identified key morphological differences between these two types of afferents, which explains how modality segregation is formed and why a rough “somatotopic map” was previously detected. Collectively, our results establish a new functional organization model for the mammalian direct dorsal column pathway and provide insight into how somatotopic and modality-based organization coexist in the central somatosensory pathway.
PMCID: PMC3818546  PMID: 24198362
6.  How do Mutations in GJB1 Cause X-linked Charcot-Marie-Tooth Disease? 
Brain research  2012;1487:198-205.
The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive weakness, atrophy, and sensory abnormalities that are most pronounced in the distal extremities. Some patients have CNS manifestations. Affected males have moderate to severe symptoms, whereas heterozygous females are usually less affected. Neurophysiology shows intermediate slowing of conduction and length-dependent axonal loss. Nerve biopsies show more prominent axonal degeneration than de/remyelination. Mutations in GJB1, the gene that encodes the gap junction (GJ) protein connexin32 (Cx32) cause CMT1X; more than 400 different mutations have been described. Many Cx32 mutants fail to form functional GJs, or form GJs with abnormal biophysical properties. Schwann cells and oligodendrocytes express Cx32, and the GJs formed by Cx32 play an important role in the homeostasis of myelinated axons. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. Effective therapies remain to be developed.
PMCID: PMC3488165  PMID: 22771394
CMT; neuropathy; connexin32; gap junctions; Schwann cells; oligodendrocytes; myelin
7.  A dominant connexin43 mutant does not have dominant effects on gap junction coupling in astrocytes 
Neuron glia biology  2011;6(4):10.1017/S1740925X11000019.
Dominant mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause oculodentodigital dysplasia (ODDD), a syndrome affecting multiple tissues, including the central nervous system (CNS). We investigated the effects of the G60S mutant, which causes a similar, dominant phenotype in mice (Gja1Jrt/+). Astrocytes in acute brain slices from Gja1Jrt/+ mice transfer sulforhodamine-B comparably to that in their wild-type (WT) littermates. Further, astrocytes and cardiomyocytes cultured from Gja1Jrt/+ mice showed a comparable transfer of lucifer yellow to those from WT mice. In transfected cells, the G60S mutant formed gap junction (GJ) plaques but not functional channels. In co-transfected cells, the G60S mutant co-immunoprecipitated with WT Cx43, but did not diminish GJ coupling as measured by dual patch clamp. Thus, whereas G60S has dominant effects, it did not appreciably reduce GJ coupling.
PMCID: PMC3848784  PMID: 21375791
ODDD; astrocytes; connexin
8.  Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2 
Brain  2013;137(1):44-56.
Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.
PMCID: PMC3891447  PMID: 24253200
childhood neuronopathy; Brown-Vialetto-Van Laere syndrome; riboflavin therapy; RFVT2; SLC52A2
9.  Prediction of Cochlear Implant Performance by Genetic Mutation: The Spiral Ganglion Hypothesis 
Hearing research  2012;292(1-2):51-58.
Up to 7% of patients with severe-to-profound deafness do not benefit from cochlear implantation. Given the high surgical implantation and clinical management cost of cochlear implantation (> $1 million lifetime cost), prospective identification of the worst performers would reduce unnecessary procedures and healthcare costs. Because cochlear implants bypass the membranous labyrinth but rely on the spiral ganglion for functionality, we hypothesize that cochlear implant (CI) performance is dictated in part by the anatomic location of the cochlear pathology that underlies the hearing loss. As a corollary, we hypothesize that because genetic testing can identify sites of cochlear pathology, it may be useful in predicting CI performance.
29 adult CI recipients with idiopathic adult-onset severe-to-profound hearing loss were studied. DNA samples were subjected to solution-based sequence capture and massively parallel sequencing using the OtoSCOPE® platform. The cohort was divided into three CI performance groups (good, intermediate, poor) and genetic causes of deafness were correlated with audiometric data to determine whether there was a gene-specific impact on CI performance.
The genetic cause of deafness was determined in 3/29 (10%) individuals. The two poor performers segregated mutations in TMPRSS3, a gene expressed in the spiral ganglion, while the good performer segregated mutations in LOXHD1, a gene expressed in the membranous labyrinth. Comprehensive literature review identified other good performers with mutations in membranous labyrinth-expressed genes; poor performance was associated with spiral ganglion-expressed genes.
Our data support the underlying hypothesis that mutations in genes preferentially expressed in the spiral ganglion portend poor CI performance while mutations in genes expressed in the membranous labyrinth portend good CI performance. Although the low mutation rate in known deafness genes in this cohort likely relates to the ascertainment characteristics (postlingual hearing loss in adult CI recipients), these data suggest that genetic testing should be implemented as part of the CI evaluation to test this association prospectively.
PMCID: PMC3461332  PMID: 22975204
Cochlear implant performance; spiral ganglion; hearing loss; genetic testing; massively parallel sequencing
10.  Neuronal cadherin (NCAD) increases sensory neurite formation and outgrowth on astrocytes 
Neuroscience letters  2012;522(2):108-112.
We examined the neurite outgrowth of sensory neurons on astrocytes following the genetic deletion of N-cadherin (NCAD). Deletion abolished immunostaining for NCAD and the other classical cadherins, indicating that NCAD is likely the only classical cadherin expressed by astrocytes. Only 38% of neurons grown on NCAD-deficient astrocytes for 24 hours produced neurites, as compared to 74% of neurons grown on NCAD-expressing astrocytes. Of the neurons that produced neurites, those grown on NCAD-deficient astrocytes had a mean total length of 378 µm, as compared to 1093 µm for neurons grown on NCAD-expressing astrocytes. Thus, the loss of NCAD greatly impairs the formation and extension neurites on astrocytes.
PMCID: PMC3784833  PMID: 22698587
astrocytes; cell adhesion; genetic deletion; NCAD; neurite outgrowth; regeneration
11.  X-linked Charcot-Marie-Tooth disease 
The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive muscle atrophy and weakness, areflexia, and variable sensory abnormalities; central nervous system manifestations occur, too. Affected males have moderate to severe symptoms, whereas heterozygous females are usually less affected. Neurophysiology shows intermediate slowing of conduction and distal axonal loss. Nerve biopsies show more prominent axonal degeneration than de/remyelination. More than 400 different mutations in GJB1, the gene that encodes the gap junction (GJ) protein connexin32 (Cx32), cause CMT1X. Many Cx32 mutants fail to form functional GJs, or form GJs with abnormal biophysical properties. Schwann cells and oligodendrocytes express Cx32, and the GJs formed by Cx32 play an important role in the homeostasis of myelinated axons. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. An effective therapy remains to be developed.
PMCID: PMC3779456  PMID: 23279425
CMT; connexin32; gap junctions; myelin; neuropathy; oligodendrocytes; Schwann cells
12.  Microtubules, Axonal Transport, and Neuropathy 
The New England journal of medicine  2011;365(24):2330-2332.
PMCID: PMC3776444  PMID: 22168648
13.  Cx32 and Cx47 mediate oligodendrocyte:astrocyte and oligodendrocyte: oligodendrocyte gap junction coupling 
Neurobiology of disease  2011;42(3):506-513.
In addition to the extensive gap junction coupling between astrocytes themselves, oligodendrocytes are thought to be exclusively coupled to astrocytes (O:A coupling) via heterotypic gap junctions composed of Cx47:Cx43 and Cx32:Cx30. We used fluorescent dyes to examine functional coupling in acute slices from the cerebra of mice lacking Cx32 and/or Cx47. In the corpus callosum, unexpectedly, oligodendrocytes appeared to be directly and exclusively coupled to other oligodendrocytes (O:O coupling), and electron microscopy revealed gap junctions between adjacent oligodendrocytes. O:O coupling was more affected in mice lacking Cx32 than in mice lacking Cx47. In the neocortex, oligodendrocytes appeared to be directly and exclusively coupled to astrocytes; Cx47, but not Cx32, was required for O:A coupling.
PMCID: PMC3773476  PMID: 21396451
Oligodendrocyte; Astrocyte; Gap junctions; Connexins
14.  Gap junctions in inherited human disorders of the central nervous system 
Biochimica et biophysica acta  2011;1818(8):2030-2047.
CNS glia and neurons express connexins, the proteins that form gap junctions in vertebrates. We review the connexins expressed by oligodendrocytes and astrocytes, and discuss their proposed physiologic roles. Of the 21 members of the human connexin family, mutations in three are associated with significant central nervous system manifestations. For each, we review the phenotype and discuss possible mechanisms of disease. Mutations in GJB1, the gene for connexin 32 (Cx32) cause the second most common form of Charcot-Marie-Tooth disease (CMT1X). Though the only consistent phenotype in CMT1X patients is a peripheral demyelinating neuropathy, CNS signs and symptoms have been found in some patients with CMT1X. Recessive mutations in GJC2, the gene for Cx47, are one cause of Pelizaeus-Merzbacher-like disease (PMLD), which is characterized by nystagmus within the first 6 months of life, cerebellar ataxia by 4 years, and spasticity by 6 years of age. MRI imaging shows abnormal myelination. A different recessive GJC2 mutation causes a form of hereditary spastic paraparesis, which is a milder phenotype than PMLD. Dominant mutations in GJA1, the gene for Cx43, cause oculodentodigital dysplasia (ODDD), a pleitropic disorder characterized by oculo-facial abnormalities including micropthalmia, microcornia and hypoplastic nares, syndactyly of the fourth to fifth fingers and dental abnormalities. Neurologic manifestations, including spasticity and gait difficulties, are often but not universally seen. Recessive GJA1 mutations cause Hallermann-Streiff syndrome, a disorder showing substantial overlap with ODDD.
PMCID: PMC3771870  PMID: 21871435
connexin; gap junction; CMT1X; PMLD1; SPG44; ODDD
15.  Reliability of the CMT neuropathy score (second version) in Charcot-Marie-Tooth disease 
The Charcot-Marie-Tooth neuropathy score (CMTNS) is a reliable and valid composite score comprising symptoms, signs, and neurophysiological tests, which has been used in natural history studies of CMT1A and CMT1X and as an outcome measure in treatment trials of CMT1A. Following an international workshop on outcome measures in Charcot-Marie-Tooth disease (CMT), the CMTNS was modified to attempt to reduce floor and ceiling effects and to standardize patient assessment, aiming to improve its sensitivity for detecting change over time and the effect of an intervention. After agreeing on the modifications made to the CMTNS (CMTNS2), three examiners evaluated 16 patients to determine inter-rater reliability; one examiner evaluated 18 patients twice within 8 weeks to determine intra-rater reliability. Three examiners evaluated 63 patients using the CMTNS and the CMTNS2 to determine how the modifications altered scoring. For inter- and intra-rater reliability, intra-class correlation coefficients (ICCs) were ≥0.96 for the CMT symptom score and the CMT examination score. There were small but significant differences in some of the individual components of the CMTNS compared with the CMTNS2, mainly in the components that had been modified the most. A longitudinal study is in progress to determine whether the CMTNS2 is more sensitive than the CMTNS for detecting change over time.
PMCID: PMC3754828  PMID: 22003934
Charcot-Marie-Tooth disease; CMT neuropathy score; reliability
16.  Butterfly genome reveals promiscuous exchange of mimicry adaptations among species 
Dasmahapatra, Kanchon K | Walters, James R. | Briscoe, Adriana D. | Davey, John W. | Whibley, Annabel | Nadeau, Nicola J. | Zimin, Aleksey V. | Hughes, Daniel S. T. | Ferguson, Laura C. | Martin, Simon H. | Salazar, Camilo | Lewis, James J. | Adler, Sebastian | Ahn, Seung-Joon | Baker, Dean A. | Baxter, Simon W. | Chamberlain, Nicola L. | Chauhan, Ritika | Counterman, Brian A. | Dalmay, Tamas | Gilbert, Lawrence E. | Gordon, Karl | Heckel, David G. | Hines, Heather M. | Hoff, Katharina J. | Holland, Peter W.H. | Jacquin-Joly, Emmanuelle | Jiggins, Francis M. | Jones, Robert T. | Kapan, Durrell D. | Kersey, Paul | Lamas, Gerardo | Lawson, Daniel | Mapleson, Daniel | Maroja, Luana S. | Martin, Arnaud | Moxon, Simon | Palmer, William J. | Papa, Riccardo | Papanicolaou, Alexie | Pauchet, Yannick | Ray, David A. | Rosser, Neil | Salzberg, Steven L. | Supple, Megan A. | Surridge, Alison | Tenger-Trolander, Ayse | Vogel, Heiko | Wilkinson, Paul A. | Wilson, Derek | Yorke, James A. | Yuan, Furong | Balmuth, Alexi L. | Eland, Cathlene | Gharbi, Karim | Thomson, Marian | Gibbs, Richard A. | Han, Yi | Jayaseelan, Joy C. | Kovar, Christie | Mathew, Tittu | Muzny, Donna M. | Ongeri, Fiona | Pu, Ling-Ling | Qu, Jiaxin | Thornton, Rebecca L. | Worley, Kim C. | Wu, Yuan-Qing | Linares, Mauricio | Blaxter, Mark L. | Constant, Richard H. ffrench | Joron, Mathieu | Kronforst, Marcus R. | Mullen, Sean P. | Reed, Robert D. | Scherer, Steven E. | Richards, Stephen | Mallet, James | McMillan, W. Owen | Jiggins, Chris D.
Nature  2012;487(7405):94-98.
The evolutionary importance of hybridization and introgression has long been debated1. We used genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation2-5 . We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,657 predicted genes for Heliconius, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organisation has remained broadly conserved since the Cretaceous, when butterflies split from the silkmoth lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, H. melpomene, H. timareta, and H. elevatus, especially at two genomic regions that control mimicry pattern. Closely related Heliconius species clearly exchange protective colour pattern genes promiscuously, implying a major role for hybridization in adaptive radiation.
PMCID: PMC3398145  PMID: 22722851
17.  The in vivo contribution of motor neuron TrkB receptors to mutant SOD1 motor neuron disease 
Human Molecular Genetics  2011;20(21):4116-4131.
Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) are widely expressed in the vertebrate nervous system and play a central role in mature neuronal function. In vitro BDNF/TrkB signaling promotes neuronal survival and can help neurons resist toxic insults. Paradoxically, BDNF/TrkB signaling has also been shown, under certain in vitro circumstances, to render neurons vulnerable to insults. We show here that in vivo conditional deletion of TrkB from mature motor neurons attenuates mutant superoxide dismutase 1 (SOD1) toxicity. Mutant SOD1 mice lacking motor neuron TrkB live a month longer than controls and retain motor function for a longer period, particularly in the early phase of the disease. These effects are subserved by slowed motor neuron loss, persistence of neuromuscular junction integrity and reduced astrocytic and microglial reactivity within the spinal cord. These results suggest that manipulation of BDNF/TrkB signaling might have therapeutic efficacy in motor neuron diseases.
PMCID: PMC3218642  PMID: 21816949
18.  High Frequency of PIK3R1 and PIK3R2 Mutations in Endometrial Cancer Elucidates a Novel Mechanism for Regulation of PTEN Protein Stability 
Cancer discovery  2011;1(2):170-185.
We demonstrate that phosphatidylinositol 3-kinase (PI3K) pathway aberrations occur in >80% of endometrioid endometrial cancers, with coordinate mutations of multiple PI3K pathway members being more common than predicted by chance. PIK3R1 (p85α) mutations occur at a higher rate in endometrial cancer than in any other tumor lineage, and PIK3R2 (p85β), not previously demonstrated to be a cancer gene, is also frequently mutated. The dominant activation event in the PI3K pathway appears to be PTEN protein loss. However, in tumors with retained PTEN protein, PI3K pathway mutations phenocopy PTEN loss, resulting in pathway activation. KRAS mutations are common in endometrioid tumors activating independent events from PI3K pathway aberrations. Multiple PIK3R1 and PIK3R2 mutations demonstrate gain of function including disruption of a novel mechanism of pathway regulation wherein p85α dimers bind and stabilize PTEN. Taken together, the PI3K pathway represents a critical driver of endometrial cancer pathogenesis and a novel therapeutic target.
PMCID: PMC3187555  PMID: 21984976
Endometrial Cancer; PTEN; PIK3CA; PIK3R1; PIK3R2
19.  Central nervous system dysfunction in a mouse model of FA2H deficiency 
Glia  2011;59(7):1009-1021.
Fatty acid 2-hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containing hydroxy fatty acid (hFA) as the N-acyl chain. Mutations in the FA2H gene cause leukodystrophy, spastic paraplegia, and neurodegeneration with brain iron accumulation. Using the Cre-lox system, we developed two types of mouse mutants, Fa2h−/− mice (Fa2h deleted in all cells by germline deletion) and Fa2hflox/flox Cnp1-Cre mice (Fa2h deleted only in oligodendrocytes and Schwann cells). We found significant demyelination, profound axonal loss, and abnormally enlarged axons in the CNS of Fa2h−/− mice at 12 months of age, while structure and function of peripheral nerves were largely unaffected. Fa2h−/− mice also exhibited histological and functional disruption in the cerebellum at 12 months of age. In a time course study, significant deterioration of cerebellar function was first detected at 7 months of age. Further behavioral assessments in water T-maze and Morris water maze tasks revealed significant deficits in spatial learning and memory at 4 months of age. These data suggest that various regions of the CNS are functionally compromised in young adult Fa2h−/− mice. The cerebellar deficits in 12-month-old Fa2hflox/flox Cnp1-Cre mice were indistinguishable from Fa2h−/−mice, indicating that these phenotypes likely stem from the lack of myelin hFA-galactolipids. In contrast, Fa2hflox/flox Cnp1-Cre mice did not show reduced performance in water maze tasks, indicating that oligodendrocytes are not involved in the learning and memory deficits found in Fa2h−/− mice. These findings provide the first evidence that FA2H has an important function outside of oligodendrocytes in the CNS.
PMCID: PMC3094470  PMID: 21491498
fatty acid 2-hydrxylase; oligodendrocytes; leukodystrophy
20.  Dominant Cx26 mutants associated with hearing loss have dominant-negative effects on wild type Cx26 
Mutations in GJB2, the gene encoding the human gap junction protein connexin26 (Cx26), cause either non-syndromic hearing loss or syndromes affecting both hearing and skin. We have investigated whether dominant Cx26 mutants can interact physically with wild type Cx26. HeLa cells stably expressing wild type Cx26 were transiently transfected to co-express nine individual dominant Cx26 mutants; six associated with non-syndromic hearing loss (W44C, W44S, R143Q, D179N, R184Q, and C202F) and three associated with hearing loss and palmoplantar keratoderma (G59A, R75Q, and R75W). All mutants co-localized and co-immunoprecipitated with wild type Cx26, indicating that they interact physically, likely by forming admixed heteromeric/heterotypic channels. Furthermore, all nine mutants inhibited the transfer of calcein in cells stably expressing Cx26, demonstrating that they each have dominant effects on wild type Cx26. Taken together, these results show that dominant-negative effects of these Cx26 mutants likely contribute to the pathogenesis of hearing loss.
PMCID: PMC3132585  PMID: 21040787
gap junctions; hearing; FRAP; immunoprecipitation; dye transfer; cochlea; disease mechanism
21.  Identification of genetic risk variants for deep vein thrombosis by multiplexed next-generation sequencing of 186 hemostatic/pro-inflammatory genes 
Next-generation DNA sequencing is opening new avenues for genetic association studies in common diseases that, like deep vein thrombosis (DVT), have a strong genetic predisposition still largely unexplained by currently identified risk variants. In order to develop sequencing and analytical pipelines for the application of next-generation sequencing to complex diseases, we conducted a pilot study sequencing the coding area of 186 hemostatic/proinflammatory genes in 10 Italian cases of idiopathic DVT and 12 healthy controls.
A molecular-barcoding strategy was used to multiplex DNA target capture and sequencing, while retaining individual sequence information. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples) and enriched for target DNA sequences. Sequencing was performed on ABI SOLiD-4 platforms. We produced > 12 gigabases of raw sequence data to sequence at high coverage (average: 42X) the 700-kilobase target area in 22 individuals. A total of 1876 high-quality genetic variants were identified (1778 single nucleotide substitutions and 98 insertions/deletions). Annotation on databases of genetic variation and human disease mutations revealed several novel, potentially deleterious mutations. We tested 576 common variants in a case-control association analysis, carrying the top-5 associations over to replication in up to 719 DVT cases and 719 controls. We also conducted an analysis of the burden of nonsynonymous variants in coagulation factor and anticoagulant genes. We found an excess of rare missense mutations in anticoagulant genes in DVT cases compared to controls and an association for a missense polymorphism of FGA (rs6050; p = 1.9 × 10-5, OR 1.45; 95% CI, 1.22-1.72; after replication in > 1400 individuals).
We implemented a barcode-based strategy to efficiently multiplex sequencing of hundreds of candidate genes in several individuals. In the relatively small dataset of our pilot study we were able to identify bona fide associations with DVT. Our study illustrates the potential of next-generation sequencing for the discovery of genetic variation predisposing to complex diseases.
PMCID: PMC3305575  PMID: 22353194
Deep vein thrombosis; venous thromboembolism; next-generation sequencing; target capture; multiplexing; FGA; rs6025; heamostateome; DVT; VTE
22.  Investigations of Caspr2, an autoantigen of encephalitis and neuromyotonia 
Annals of neurology  2011;69(2):303-311.
To report clinical and immunological investigations of contactin-associated protein-like 2 (Caspr2), an autoantigen of encephalitis and peripheral nerve hyperexcitability (PNH) previously attributed to voltage-gated potassium channels (VGKC).
Clinical analysis of patients with encephalitis, PNH, or both. Immunoprecipitation and mass spectrometry were used to identify the antigen and to develop an assay with Caspr2-expressing cells. Immunoabsorption with Caspr2 and comparative immunostaining of brain and peripheral nerve of wild-type and Caspr2-null mice were used to assess antibody specificity.
Using Caspr2-expressing cells, antibodies were identified in 8 patients but not in 140 patients with several types of autoimmune or viral encephalitis, PNH, or mutations of the Caspr2-encoding gene. Patients’ antibodies reacted with brain and peripheral nerve in a pattern that co-localized with Caspr2. This reactivity was abrogated after immunoabsorption with Caspr2 and was absent in tissues from Caspr2-null mice. Of the 8 patients with Caspr2 antibodies, 7 had encephalopathy or seizures, 5 neuropathy or PNH, and 1 isolated PNH. Three patients had also myasthenia gravis, bulbar weakness, or symptoms that initially suggested motor neuron disease. None of the patients had active cancer; 7 responded to immunotherapy and were healthy or only mildly disabled at last follow-up (median 8 months, range 6–84).
Caspr2 is an autoantigen of encephalitis and PNH previously attributed to VGKC antibodies. The occurrence of other autoantibodies may result in a complex syndrome that at presentation could be mistaken for a motor neuron disorder. Recognition of this disorder is important because it responds to immunotherapy.
PMCID: PMC3059252  PMID: 21387375
autoimmune; encephalitis; seizures; neuromyotonia; peripheral nerve hyperexcitability; VGKC; Caspr2
23.  The debut of a rational treatment for an inherited neuropathy? 
The Journal of Clinical Investigation  2011;121(12):4624-4627.
Hereditary neuropathies are common neurological conditions characterized by progressive loss of motor and/or sensory function. There are no effective treatments. Among the many causes of hereditary neuropathies are dominant mutations in serine palmitoyltransferase, long chain base subunit 1 (SPTLC1), which cause hereditary sensory and autonomic neuropathy type 1 (HSAN1). By incorporating l-alanine in place of l-serine, the mutant HSAN1–associated serine palmitoyltransferase generates deoxysphingolipids, which are thought to be neurotoxic. In this issue of the JCI, Garofalo and colleagues report that oral l-serine reverses the accumulation of deoxysphingolipids in humans with HSAN1 and in a transgenic mouse model. As oral l-serine reduces the severity of neuropathy in the mouse model of HSAN1, these data suggest a rational candidate therapy for this devastating condition.
PMCID: PMC3226011  PMID: 22045569
24.  Axonal Pathology Precedes Demyelination in a Mouse Model of X-Linked Demyelinating/ Type I Charcot-Marie Tooth (CMT1X) Neuropathy 
X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the gene that encodes the gap junction protein connexin32 (Cx32). Cx32 is expressed by myelinating Schwann cells and forms gap junctions in non-compact myelin areas but axonal involvement is more prominent in X-linked compared to other forms of demyelinating Charcot-Marie-Tooth disease. To clarify the cellular and molecular mechanisms of axonal pathology in CMT1X, we studied Gjb1-null mice at early stages (i.e. 2- to 4-month-old) of the neuropathy, when there is minimal or no demyelination. The diameters of large myelinated axons were progressively reduced in Gjb1-null mice compared to those in wild type littermates. Furthermore, neurofilaments were relatively more dephosphorylated and more densely packed starting at 2 months of age. Increased expression of β-amyloid precursor protein, a marker of axonal damage, was also detected in Gjb1-null nerves. Finally, fast axonal transport, assayed by sciatic nerve ligation experiments, was slower in distal axons of Gjb1-null vs. wild type animals with reduced accumulation of synaptic vesicle-associated proteins. These findings demonstrate that axonal abnormalities including impaired cytoskeletal organization and defects in axonal transport precede demyelination in this mouse model of CMT1-X.
PMCID: PMC3034224  PMID: 20720503
Axonal degeneration; Axonal transport; Charcot-Marie Tooth; Connexin32; Cx32; Gap junctions; Neurofilaments
25.  A genetic map of Xenopus tropicalis 
Developmental Biology  2011;354(1-2):1-8.
We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.
Research highlights
► A genetic map of 2886 Simple Sequence Length Polymorphisms for Xenopus tropicalis. ► 10 major linkage groups corresponding to the 10 chromosomes, plus 4 minor linkage groups. ► Linkage groups are cytogenetically mapped to chromosomes by Fluorescence In Situ Hybridization.
PMCID: PMC3098391  PMID: 21458440
Xenopus; X. tropicalis; Genetic map; Genome; Simple sequence length polymorphism

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