Towards complete and error-free genome assemblies of all vertebrate species

The Vertebrate Genomes Project aims to provide reference-quality assemblies for all 70,000 extant vertebrate species. The long-read sequencing team was one of three sequencing hubs and contributed chromosome-level assemblies for a number of vertebrate species for phase 1. In this first phase, 16 new assemblies from six major vertebrate lineages have been contributed, which established the groundwork for expansion to produce genome assemblies representing the remaining vertebrate orders over the coming years. These reference-quality genome assemblies reveal key genetic distinctions between the vertebrate lineages at the level of chromosome evolution and gene selection and will eventually help to preserve the genetic information of life.


Six reference-quality genomes reveal evolution of bat adaptations

The Bat1K consortium aims to sequence all ~1,300 bat species and provide chromosome-level assemblies to the public domain. The long-read sequencing team sequenced and assembled the first 6 bat species, which were published in the Bat1K’s 2020 flagship paper. This paper release high-quality, high-contiguity, chromosome-scale assemblies for 6 bat families, revealing the genetic basis for evolution of flight in bats among mammals, shed some light on the specific immune system of bats, and the origins of viral resistance. Phase 2 of the Bat1K efforts is currently underway, aiming to provide sequences representing all 21 bat families.

* joint first author # joint corresponding author

Felipe Mora-Bermúdez, Philipp Kanis, Dominik Macak, Jula Peters, Ronald Naumann, Lei Xing, Mihail Sarov, Sylke Winkler, Christina Eugster Oegema, Christiane Haffner, Pauline Wimberger, Stephan Riesenberg, Tomislav Maricic, Wieland Huttner, Svante Pääbo
Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.
Sci Adv, 8(30) Art. No. eabn7702 (2022)
Open Access DOI
Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

Kang Du, Martin Pippel, Susanne Kneitz, Romain Feron, Irene da Cruz, Sylke Winkler, Brigitta Wilde, Edgar G Avila Luna, Gene Myers, Yann Guiguen, Constantino Macias Garcia, Manfred Schartl
Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation.
Genome Res, 32(3) 583-594 (2022)
Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.

Juliana G. Roscito#, Katrin Sameith, Bogdan Kirilenko, Nikolai Hecker, Sylke Winkler, Andreas Dahl, Miguel Trefaut Rodrigues, Michael Hiller#
Convergent and lineage-specific genomic differences in limb regulatory elements in limbless reptile lineages.
Cell Rep, 38(3) Art. No. 110280 (2022)
Open Access DOI
Loss of limbs evolved many times in squamate reptiles. Here we investigated the genomic basis of convergent limb loss in reptiles. We sequenced the genomes of a closely related pair of limbless-limbed gymnophthalmid lizards and performed a comparative genomic analysis including five snakes and the limbless glass lizard. Our analysis of these three independent limbless lineages revealed that signatures of shared sequence or transcription factor binding site divergence in individual limb regulatory elements are generally rare. Instead, shared divergence occurs more often at the level of signaling pathways, involving different regulatory elements associated with the same limb genes (such as Hand2 or Hox) and/or patterning mechanisms (such as Shh signaling). Interestingly, although snakes are known to have mutations in the Shh ZRS limb enhancer, this enhancer lacks relevant mutations in limbless lizards. Thus, different mechanisms could contribute to limb loss, and there are likely multiple evolutionary paths to limblessness in reptiles.

Marlena Stadtmüller, Alexa Laubner, Fabian Rost, Sylke Winkler, Eva Patrasová, Lenka Šimůnková, Susanne Reinhardt, Johanna Beil, Alexander Dalpke, Buqing Yi
Emergence and spread of a sub-lineage of SARS-CoV-2 Alpha variant B.1.1.7 in Europe, and with further evolution of spike mutation accumulations shared with the Beta and Gamma variants.
Virus Evol, 8(1) Art. No. veac010 (2022)
Open Access DOI
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution plays a significant role in shaping the dynamics of the coronavirus disease 2019 pandemic. To monitor the evolution of SARS-CoV-2 variants, through international collaborations, we performed genomic epidemiology analyses on a weekly basis with SARS-CoV-2 samples collected from a border region between Germany, Poland, and the Czech Republic in a global background. For identified virus mutant variants, active viruses were isolated and functional evaluations were performed to test their replication fitness and neutralization sensitivity against vaccine-elicited serum neutralizing antibodies. Thereby we identified a new B.1.1.7 sub-lineage carrying additional mutations of nucleoprotein G204P and open-reading-frame-8 K68stop. Of note, this B.1.1.7 sub-lineage is the predominant B.1.1.7 variant in several European countries such as Czech Republic, Austria, and Slovakia. The earliest samples belonging to this sub-lineage were detected in November 2020 in a few countries in the European continent, but not in the UK. We have also detected its further evolution with extra spike mutations D138Y and A701V, which are signature mutations shared with the Gamma and Beta variants, respectively. Antibody neutralization assay of virus variant isolations has revealed that the variant with extra spike mutations is 3.2-fold less sensitive to vaccine-elicited antibodies as compared to the other B.1.1.7 variants tested, indicating potential for immune evasion, but it also exhibited reduced replication fitness, suggesting lower transmissibility. The wide spread of this B.1.1.7 sub-lineage was related to the pandemic waves in early 2021 in various European countries. These findings about the emergence, spread, evolution, infection, and transmission abilities of this B.1.1.7 sub-lineage add to our understanding about the pandemic development in Europe and highlight the importance of international collaboration on virus mutant surveillance.

Lina Muhandes, Maria Chapsa, Martin Pippel, Rayk Behrendt, Yan Ge, Andreas Dahl, Buqing Yi, Alexander Dalpke, Sylke Winkler, Michael Hiller, Sebastien Boutin, Stefan Beissert, Rolf Jessberger, Padraic G Fallon, Axel Roers
Low Threshold for Cutaneous Allergen Sensitization but No Spontaneous Dermatitis or Atopy in FLG-Deficient Mice.
J Invest Dermatol, 141(11) 2611-2619 (2021)
Open Access DOI
Loss of FLG causes ichthyosis vulgaris. Reduced FLG expression compromises epidermal barrier function and is associated with atopic dermatitis, allergy, and asthma. The flaky tail mouse harbors two mutations that affect the skin barrier, Flgft, resulting in hypomorphic FLG expression, and Tmem79ma, inactivating TMEM79. Mice defective only for TMEM79 featured dermatitis and systemic atopy, but also Flgft/ft BALB/c congenic mice developed eczema, high IgE, and spontaneous asthma, suggesting that FLG protects from atopy. In contrast, a targeted Flg-knockout mutation backcrossed to BALB/c did not result in dermatitis or atopy. To resolve this discrepancy, we generated FLG-deficient mice on pure BALB/c background by inactivating Flg in BALB/c embryos. These mice feature an ichthyosis phenotype, barrier defect, and facilitated percutaneous sensitization. However, they do not develop dermatitis or atopy. Whole-genome sequencing of the atopic Flgft BALB/c congenics revealed that they were homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that FLG deficiency does not cause atopy in mice, in line with lack of atopic disease in a fraction of patients with ichthyosis vulgaris carrying two Flg null alleles. However, the absence of FLG likely promotes and modulates dermatitis caused by other genetic barrier defects.

Stefano Suzzi, Reiner Ahrendt, Stefan Hans, Svetlana A Semenova, Avinash Chekuru, Paul Wirsching, Volker Kroehne, Saygın Bilican, Shady Sayed, Sylke Winkler, Sandra Spieß, Anja Machate, Jan Kaslin, Pertti Panula, Michael Brand
Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.
PLoS Genet, 17(9) Art. No. 1009794 (2021)
Open Access DOI
LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

Buqing Yi, Ansgar Poetsch, Marlena Stadtmüller, Fabian Rost, Sylke Winkler, Alexander Dalpke
Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany in 2020: insights into the cause of the second wave.
Epidemiol Infect, 149 Art. No. e177 (2021)
Open Access DOI
In Germany, Eastern regions had a mild first wave of coronavirus disease 2019 (COVID-19) from March to May 2020, but were badly hit by a second wave later in autumn and winter. It is unknown how the second wave was initiated and developed in Eastern Germany where the number of COVID-19 cases was close to zero in June and July 2020. We used genomic epidemiology to investigate the dynamic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage development across the first and second waves in Eastern Germany. With detailed phylogenetic analyses we could show that SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with several new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August and October 2020. The results indicate that the major driving force behind the second wave was the introduction of new variants.

Giulio Formenti#, Arang Rhie, Jennifer Balacco, Bettina Haase, Jacquelyn Mountcastle, Olivier Fedrigo, Samara Brown, Marco Rosario Capodiferro, Farooq O Al-Ajli, Roberto Ambrosini, Peter Houde, Sergey Koren, Karen Oliver, Michelle Smith, Jason Skelton, Emma Betteridge, Jale Dolucan, Craig Corton, Iliana Bista, James Torrance, Alan Tracey, Jonathan Wood, Marcela Uliano-Silva, Kerstin Howe, Shane A McCarthy, Sylke Winkler, Woori Kwak, Jonas Korlach, Arkarachai Fungtammasan, Daniel Fordham, Vania Costa, Simon Mayes, Matteo Chiara, David S Horner, Eugene W Myers, Richard Durbin, Alessandro Achilli, Edward L Braun, Adam M Phillippy, Erich D Jarvis#, Erich D null
Complete vertebrate mitogenomes reveal widespread repeats and gene duplications.
Genome Biol, 22(1) Art. No. 120 (2021)
Open Access DOI
Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly.

Arang Rhie, Shane A McCarthy, Olivier Fedrigo, Joana Damas, Giulio Formenti, Sergey Koren, Marcela Uliano-Silva, William Chow, Arkarachai Fungtammasan, Juwan Kim, Chul Lee, Byung June Ko, Mark Chaisson, Gregory L Gedman, Lindsey J Cantin, Francoise Thibaud-Nissen, Leanne Haggerty, Iliana Bista, Michelle Smith, Bettina Haase, Jacquelyn Mountcastle, Sylke Winkler, Sadye Paez, Jonathon Howard, Sonja Vernes, Tanya M Lama, Frank Grutzner, Wesley C Warren, Christopher N Balakrishnan, Dave Burt, Julia M George, Matthew T Biegler, David Iorns, Andrew Digby, Daryl Eason, Bruce Robertson, Taylor Edwards, Mark Wilkinson, George Turner, Axel Meyer, Andreas F Kautt, Paolo Franchini, H William Detrich, Hannes Svardal, Maximilian Wagner, Gavin J P Naylor, Martin Pippel, Milan Malinsky, Mark Mooney, Maria Simbirsky, Brett T Hannigan, Trevor Pesout, Marlys Houck, Ann Misuraca, Sarah B Kingan, Richard J Hall, Zev Kronenberg, Ivan Sović, Christopher Dunn, Zemin Ning, Alex R Hastie, Joyce Lee, Siddarth Selvaraj, Richard E Green, Nicholas H Putnam, Ivo Gut, Jay Ghurye, Erik Garrison, Ying Sims, Joanna Collins, Sarah Pelan, James Torrance, Alan Tracey, Jonathan Wood, Robel E Dagnew, Dengfeng Guan, Sarah E London, David F Clayton, Claudio V Mello, Samantha R Friedrich, Peter V Lovell, Ekaterina Osipova, Farooq O Al-Ajli, Simona Secomandi, Heebal Kim, Constantina Theofanopoulou, Michael Hiller, Yang Zhou, Robert S Harris, Kateryna D Makova, Paul Medvedev, Jinna Hoffman, Patrick Masterson, Karen Clark, Fergal Martin, Kerstin Howe#, Paul Flicek, Brian Walenz, Woori Kwak, Hiram Clawson, Mark Diekhans, Luis Nassar, Benedict Paten, Robert H S Kraus, Andrew J Crawford, M Thomas P Gilbert, Guojie Zhang, Byrappa Venkatesh, Robert F Murphy, Klaus-Peter Koepfli, Beth Shapiro, Warren E Johnson, Federica Di Palma, Tomas Marques-Bonet, Emma Teeling, Tandy Warnow, Jennifer Marshall Graves, Oliver A Ryder, David Haussler, Stephen J O'Brien, Jonas Korlach, Harris A Lewin, Kerstin Howe#, Eugene W Myers#, Richard Durbin#, Adam M Phillippy#, Erich D Jarvis#
Towards complete and error-free genome assemblies of all vertebrate species.
Nature, 592(7856) 737-746 (2021)
Open Access DOI
High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Sarita Hebbar✳︎, Malte Lehmann✳︎, Sarah Behrens, Catrin Hälsig, Weihua Leng, Michaela Yuan, Sylke Winkler, Elisabeth Knust
Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila.
Biol Open, 10(1) Art. No. bio052332 (2021)
Open Access DOI
Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease.This paper has an associated First Person interview with the co-first authors of the article.

David Jebb✳︎, Zixia Huang✳︎, Martin Pippel✳︎, Graham M Hughes, Ksenia Lavrichenko, Paolo Devanna, Sylke Winkler, Lars S Jermiin, Emilia C Skirmuntt, Aris Katzourakis, Lucy Burkitt-Gray, David A Ray, Kevin F. Sullivan, Juliana G. Roscito, Bogdan Kirilenko, Liliana M Dávalos, Angelique P Corthals, Megan L Power, Gareth Jones, Roger D Ransome, Dina K N Dechmann, Andrea G Locatelli, Sébastien J Puechmaille, Olivier Fedrigo, Erich D Jarvis, Michael Hiller#, Sonja Vernes#, Eugene W Myers#, Emma Teeling#
Six reference-quality genomes reveal evolution of bat adaptations.
Nature, 583(7817) 578-584 (2020)
Open Access DOI
Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our 'Tool to infer Orthologs from Genome Alignments' (TOGA) software with de novo and homology gene predictions as well as short- and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease1.

Irene Adrian-Kalchhauser, Anders Blomberg, Tomas Larsson, Zuzana Musilova, Claire R Peart, Martin Pippel, Monica Hongroe Solbakken, Jaanus Suurväli, Jean-Claude Walser, Joanna Yvonne Wilson, Magnus Alm Rosenblad, Demian Burguera, Silvia Gutnik, Nico Michiels, Mats Töpel, Kirill Pankov, Siegfried Schloissnig, Sylke Winkler
The round goby genome provides insights into mechanisms that may facilitate biological invasions.
BMC Biol, 18(1) Art. No. 11 (2020)
Open Access DOI
The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success.

Satu Kujawski✳︎, Catia Crespo✳︎, Marta Luz, Michaela Yuan, Sylke Winkler, Elisabeth Knust
Loss of Crb2b-lf leads to anterior segment defects in old zebrafish.
Biol Open, 9(2) Art. No. bio.047555 (2020)
Open Access DOI
Defects in the retina or the anterior segment of the eye lead to compromised vision and affect millions of people. Understanding how these ocular structures develop and are maintained is therefore of paramount importance. The maintenance of proper vision depends, among others, on the function of genes controlling apico-basal polarity. In fact, mutations in polarity genes are linked to retinal degeneration in several species, including human. Here we describe a novel zebrafish crb2b allele (crb2b e40 ), which specifically affects the crb2b long isoform. crb2b e40 mutants are viable and display normal ocular development. However, old crb2b e40 mutant fish develop multiple defects in structures of the anterior segment, which includes the cornea, the iris and the lens. Phenotypes are characterised by smaller pupils due to expansion of the iris and tissues of the iridocorneal angle, an increased number of corneal stromal keratocytes, an abnormal corneal endothelium and an expanded lens capsule. These findings illustrate a novel role for crb2b in the maintenance of the anterior segment and hence add an important function to this polarity regulator, which may be conserved in other vertebrates, including humans.

Martin Pippel✳︎, David Jebb✳︎, Franziska Patzold, Sylke Winkler, Heiko Vogel, Gene Myers, Michael Hiller#, Anna K Hundsdoerfer#
A highly contiguous genome assembly of the bat hawkmoth Hyles vespertilio (Lepidoptera: Sphingidae).
GigaScience, 9(1) Art. No. giaa001 (2020)
Open Access DOI
Adapted to different ecological niches, moth species belonging to the Hyles genus exhibit a spectacular diversity of larval color patterns. These species diverged ∼7.5 million years ago, making this rather young genus an interesting system to study a wide range of questions including the process of speciation, ecological adaptation, and adaptive radiation.

Juliana G. Roscito, Katrin Sameith, Martin Pippel, Kees-Jan Francoijs, Sylke Winkler, Andreas Dahl, Georg Papoutsoglou, Gene Myers, Michael Hiller
The genome of the tegu lizard Salvator merianae: combining Illumina, PacBio, and optical mapping data to generate a highly contiguous assembly.
GigaScience, 7(12) Art. No. giy141 (2018)
Open Access PDF DOI
Reptiles are a species-rich group with great phenotypic and life history diversity but are highly underrepresented among the vertebrate species with sequenced genomes.

Samir Vaid, J Gray Camp, Lena Hersemann, Christina Eugster Oegema, Anne-Kristin Heninger, Sylke Winkler, Holger Brandl, Mihail Sarov, Barbara Treutlein, Wieland Huttner#, Takashi Namba#
A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.
Development, 145(20) Art. No. dev169276 (2018)
A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.

Virag Sharma, Thomas Lehmann, Heiko Stuckas, Liane Funke, Michael Hiller
Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals.
PLoS Biol, 16(6) Art. No. e2005293 (2018)
Open Access PDF DOI
Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These "molecular vestiges" provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

Marta Florio✳︎, Michael Heide✳︎, Anneline Pinson, Holger Brandl, Mareike Albert, Sylke Winkler, Pauline Wimberger, Wieland B. Huttner#, Michael Hiller#
Evolution and cell-type specificity of human-specific genes preferentially expressed in progenitors of fetal neocortex.
Elife, 7 Art. No. e32332 (2018)
Open Access PDF DOI
Understanding the molecular basis that underlies the expansion of the neocortex during primate, and notably human, evolution requires the identification of genes that are particularly active in the neural stem and progenitor cells of the developing neocortex. Here, we have used existing transcriptome datasets to carry out a comprehensive screen for protein-coding genes preferentially expressed in progenitors of fetal human neocortex. We show that fifteen human-specific genes exhibit such expression, and many of them evolved distinct neural progenitor cell-type expression profiles and levels compared to their ancestral paralogs. Functional studies on one such gene,NOTCH2NL, demonstrate its ability to promote basal progenitor proliferation in mice. An additional 35 human genes with progenitor-enriched expression are shown to have orthologs only in primates. Our study provides a resource of genes that are promising candidates to exert specific, and novel, roles in neocortical development during primate, and notably human, evolution.

Markus Grohme✳︎, Siegfried Schloissnig✳︎, Andrei Rozanski, Martin Pippel, George Robert Young, Sylke Winkler, Holger Brandl, Ian Henry, Andreas Dahl, Sean Powell, Michael Hiller, Eugene Myers, Jochen Rink
The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms.
Nature, 554(7690) 56-61 (2018)
Open Access PDF DOI
The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.

Sergej Nowoshilow, Siegfried Schloissnig, Jifeng Fei, Andreas Dahl, Andy W C Pang, Martin Pippel, Sylke Winkler, Alex R Hastie, George Young, Juliana G. Roscito, Francisco Falcon, Dunja Knapp, Sean Powell, Alfredo Cruz, Han Cao, Bianca Habermann, Michael Hiller, Elly M. Tanaka, Eugene W Myers
The axolotl genome and the evolution of key tissue formation regulators.
Nature, 554(7690) 50-55 (2018)
Open Access PDF DOI
Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/-and Pax7-/-mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

V Albrecht, C Zweiniger, Vineeth Surendranath, Karl Lang, G Schöfl, Andreas Dahl, Sylke Winkler, V Lange, I Böhme, A H Schmidt
Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles.
HLA, 90(2) 79-87 (2017)
Open Access DOI
The high-throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5'- to 3'-UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory - European Nucleotide Archive (EMBL-ENA) and the IPD-IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real-time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full-length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi-automated web service TypeLoader, to aid in the submission of sequences to the EMBL-ENA and the IPD-IMGT/HLA Database. In the IPD-IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full-length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.

R Urrego, Sandra Milena Bernal-Ulloa, N A Chavarría, E Herrera-Puerta, Andrea Lucas-Hahn, D Herrmann, Sylke Winkler, Dorit Pache, H Niemann, N Rodriguez-Osorio
Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.
Zygote, 25(2) 131-140 (2017)
Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Irene Adrian-Kalchhauser, Ola Svensson, Verena E Kutschera, Magnus Alm Rosenblad, Martin Pippel, Sylke Winkler, Siegfried Schloissnig, Anders Blomberg, Patricia Burkhardt-Holm
The mitochondrial genome sequences of the round goby and the sand goby reveal patterns of recent evolution in gobiid fish.
BMC Genomics, 18(1) Art. No. 177 (2017)
Open Access PDF DOI
Vertebrate mitochondrial genomes are optimized for fast replication and low cost of RNA expression. Accordingly, they are devoid of introns, are transcribed as polycistrons and contain very little intergenic sequences. Usually, vertebrate mitochondrial genomes measure between 16.5 and 17 kilobases (kb).

Stephanie Spannl✳︎, Alexandra Kumichel✳︎, Sarita Hebbar, Katja Kapp, Marcos Gonzalez-Gaitan, Sylke Winkler, Rosana Blawid, Gregor Jessberger, Elisabeth Knust
The Crumbs_C isoform of Drosophila shows tissue- and stage-specific expression and prevents light-dependent retinal degeneration.
Biol Open, 6(2) 165-175 (2017)
Open Access PDF DOI
Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Nereo Kalebic, Elena Taverna, Stefania Tavano, Fong Kuan Wong, Dana Suchold, Sylke Winkler, Wieland B. Huttner, Mihail Sarov
CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.
EMBO Rep, 17(3) 338-348 (2016)
Open Access PDF DOI
We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

Sandra Milena Bernal-Ulloa, Julia Heinzmann, Doris Herrmann, Klaus-Gerd Hadeler, Patrick Aldag, Sylke Winkler, Dorit Pache, Ulrich Baulain, Andrea Lucas-Hahn, Heiner Niemann
Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle.
PLoS ONE, 11(2) Art. No. e0150264 (2016)
Open Access PDF DOI
High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.

Sylke Winkler, K. Linke, Nicola Gscheidel, M. Meyer, D. Krabel
PCR-based detection of single sequence variants from a natural collection of the non-model tree species European Aspen Populus tremula (L.)
Silvae Genetica, 64(5-6) 259-269 (2015)
Open Access
In the present study we present and discuss the identification of species-specific SNPs to rule out any experimental influence of species specific primer design (Populus tremula vs. the closely related model-species Populus trichocarpa) on the detectability of SNPs. Applying a species-optimized method, partial sequences of 14 genes involved in xylem cell development, xylogenesis, pectin formation, and drought stress reaction were analyzed at the genomic level. About 3 Mb of sequence information were generated by Sanger sequencing technology and 258 sequence variants were identified. 15 out of these represent insertions/deletions located exclusively in non coding regions and the remaining 243 are SNPs found in coding and non-coding regions of candidate genes. The introduction of a species-specific SNP detection pipeline will help to detect nucleotide variants in P. tremula and to conduct association mapping in natural P tremula populations.

Pooja Kumari, Patrick C Gilligan, Shimin Lim, Long Duc Tran, Sylke Winkler, Robin Philp, Karuna Sampath
An essential role for maternal control of Nodal signaling.
Elife, 2 Art. No. e00683 (2013)
Open Access DOI
Growth factor signaling is essential for pattern formation, growth, differentiation, and maintenance of stem cell pluripotency. Nodal-related signaling factors are required for axis formation and germ layer specification from sea urchins to mammals. Maternal transcripts of the zebrafish Nodal factor, Squint (Sqt), are localized to future embryonic dorsal. The mechanisms by which maternal sqt/nodal RNA is localized and regulated have been unclear. Here, we show that maternal control of Nodal signaling via the conserved Y box-binding protein 1 (Ybx1) is essential. We identified Ybx1 via a proteomic screen. Ybx1 recognizes the 3' untranslated region (UTR) of sqt RNA and prevents premature translation and Sqt/Nodal signaling. Maternal-effect mutations in zebrafish ybx1 lead to deregulated Nodal signaling, gastrulation failure, and embryonic lethality. Implanted Nodal-coated beads phenocopy ybx1 mutant defects. Thus, Ybx1 prevents ectopic Nodal activity, revealing a new paradigm in the regulation of Nodal signaling, which is likely to be conserved. DOI:

Sylke Winkler, Nicola Gscheidel, Michael Brand
Mutant generation in vertebrate model organisms by TILLING.
Methods Mol Biol, 770 475-504 (2011)
TILLING (Targeting Induced Local Lesions IN Genomes) is a popular reverse genetic approach that has been successfully applied in several genetic model organisms such as zebrafish, rat, Drosophila, Arabidopsis, or medaka. In contrast to classical targeted knockout technologies that work in mice by directly targeting a gene of interest, TILLING follows an indirect strategy. The first step of the TILLING pipeline is the generation of a TILLING library that consists of large numbers of mutagenized individuals. In a second step, these individuals are screened for mutations in any gene of interest. Screening is performed by PCR amplification of specific exons from each individual of a library followed by mutation detection. This could be done, for example, by direct re-sequencing of PCR fragments or alternatively, by CEL1 endonuclease-mediated mutation discovery. Individuals carrying potentially deleterious point mutations are isolated from the library and mutant lines are established. TILLING allows the identification of a whole range of point mutations, covering nonsense, splice site, and missense mutations in only one screening round, because the generation of mutations by mutagenesis as well as the screening tools is not biased. Potential knockout mutations are initially the mutations of choice, but TILLING screens can also be used to isolate allelic series of point mutations ranging from complete null phenotypes to hypomorphic or even dominant-negative or conditional alleles. These allelic series can be helpful for a comprehensive functional analysis of a gene of interest. TILLING is applicable to any kind of genetically tractable model organism, as long as this model organism is amenable to chemical mutagenesis, and genomic sequence information for a gene of interest is available. This chapter describes the design and pipeline of a TILLING facility as we are currently operating it for zebrafish in Dresden. Protocols for mutation detection by direct re-sequencing are described in detail. However, alternatives to this pipeline do exist and will be mentioned briefly.

Elena Quesada-Hernández, Luca Caneparo, Sylvia Schneider, Sylke Winkler, Michael Liebling, Scott E Fraser, Carl-Philipp Heisenberg
Stereotypical cell division orientation controls neural rod midline formation in zebrafish.
Curr Biol, 20(21) 1966-1972 (2010)
The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body-axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Here we show that SDO is required for formation of the neural rod midline during neurulation but dispensable for elongation of the body axis during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the noncanonical Wnt receptor Frizzled 7 (Fz7) and that interfering with cell division orientation leads to severe defects in neural rod midline formation but not body-axis elongation. These findings suggest a novel function for Fz7-controlled cell division orientation in neural rod midline formation during neurulation.

Radoslaw K Ejsmont, Mihail Sarov, Sylke Winkler, Kamil A Lipinski, Pavel Tomancák
A toolkit for high-throughput, cross-species gene engineering in Drosophila.
Nat Methods, 6(6) 435-437 (2009)
We generated two complementary genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura that permit seamless modification of large genomic clones by high-throughput recombineering and direct transgenesis. The fosmid transgenes recapitulated endogenous gene expression patterns. These libraries, in combination with recombineering technology, will be useful to rescue mutant phenotypes, allow imaging of gene products in living flies and enable systematic analysis and manipulation of gene activity across species.

John W Hawes, Kevin L Knudtson, Helaman Escobar, George S Grills, Timothy C Hunter, Emily Jackson-Machelski, Heather Lin, David S Needleman, Rashmi Pershad, Glenis J. Wiebe
Evaluation of methods for sequence analysis of highly repetitive DNA templates.
J Biomol Tech, 17(2) 138-144 (2006)
The DNA Sequencing Research Group (DSRG) of the ABRF conducted a study to assess the ability of DNA sequencing core facilities to successfully sequence a set of well-defined templates containing difficult repeats. The aim of this study was to determine whether repetitive templates could be sequenced accurately by using equipment and chemistries currently utilized in participating sequencing laboratories. The effects of primer and template concentrations, sequencing chemistries, additives, and instrument formats on the ability to successfully sequence repeat elements were examined. The first part of this study was an analysis of the results of 361 chromatograms from participants representing 40 different laboratories who attempted to sequence a panel of difficult-to-sequence templates using their best in-house protocols. The second part of this study was a smaller multi-laboratory evaluation of a single robust protocol with the same panel of templates. This study provides a measure of the potential success of different approaches to sequencing across homopolymer tracts and repetitive elements.

Oliver Bartsch, Stefanie Schmidt, Marion Richter, Susanne Morlot, Eva Seemanová, Glenis J. Wiebe, Sasan Rasi
DNA sequencing of CREBBP demonstrates mutations in 56% of patients with Rubinstein-Taybi syndrome (RSTS) and in another patient with incomplete RSTS.
Hum Genet, 117(5) 485-493 (2005)
Rubinstein-Taybi syndrome (RSTS) is a distinct dominant disorder characterized by short stature, typical face, broad angulated thumbs and halluces, and mental retardation. The RSTS can be caused by chromosomal microdeletions and molecular mutations in the CREBBP gene; however, relatively few mutations have been reported to date. Here, we aimed to determine the rate of point mutations and other small molecular lesions in true RSTS and possible mild variants, by using genomic DNA sequencing. A consecutive series of patients including 17 patients from our previous study was investigated. We identified 19 causative mutations of CREBBP in a total of 45 patients representing three different diagnostic groups: (a) 17 mutations in 30 patients with unequivocal RSTS (detection rate 56.6%), (b) two mutations in eight patients with features suggestive of RSTS ("moderate or incomplete RSTS", detection rate 25%), and (c) no mutation in seven patients with undiagnosed syndromes and isolated features of RSTS. In general, the mutations were distributed without hot spots and most were unique; however, three recurrent mutations (R370X, R1664H, and N1978S) were identified. Furthermore, we detected 15 different intragenic polymorphisms, including two non-synonymous coding polymorphisms, L551I and Q2208H. We report not only the highest detection rate (56.6%) of CREBBP mutations in patients with RSTS to date, but also the second missense mutation (N1978S) in a patient with moderate or incomplete RSTS. Previous studies have identified cytogenetic deletions in the CREBBP gene in eight to 12% of patients and very recently, Roelfsema et al. reported EP300 gene mutations in three of 92 (3.3%) patients with either true RSTS or different syndromes resembling RSTS. Our 56.6% detection rate of molecular mutations in CREBBP in patients with unequivocal RSTS supports the new concept that RSTS is a genetically heterogeneous disorder and furthermore, indicates that RSTS may be caused by gene/s other than CREBBP in up to 30% of cases.

Sylke Winkler, Anja Schwabedissen, Dana Backasch, Christian Bökel, Claudia Seidel, Stefanie Bönisch, Maximilian Fürthauer, Antje Kuhrs, Laura Cobreros, Michael Brand, Marcos González-Gaitán
Target-selected mutant screen by TILLING in Drosophila.
Genome Res, 15(5) 718-723 (2005)
The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized mutations have been generated in around 6000 of the approximately 15,000 annotated fly genes, of which around one-third are essential for viability. To obtain mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based on the large-scale detection of point mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations in three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded an average of one mutation every 156 kb. For an essential gene tested, five of 25 mutations turned out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.

Minna H Pöyhönen, Maarit M Peippo, Leena K Valanne, Kirsti E Kuokkanen, Susanna M Koskela, Oliver Bartsch, Sasan Rasi, Glenis J. Wiebe, Marketta Kähkönen, Helena A Kääriäinen
Hypertrichosis, hyperkeratosis, abnormal corpus callosum, mental retardation and dysmorphic features in three unrelated females.
Clin Dysmorphol, 13(2) 85-90 (2004)
We report three unrelated patients with hypertrichosis, mild to moderate mental retardation, and dysmorphic facial features including low anterior hairline, thick arched eyebrows, nose with broad tip and columella below alae nasi, short philtrum, thick drooping lower lip and simple posteriorly rotated ears. They also had rough skin with hyperkeratotic plaques. Feet and finger tips were broad. All of them had personality problems like aggressiveness, stubborn temperament or tendency to withdraw. Brain MRI showed thick and short corpus callosum. We believe that these patients represent a new syndrome of unknown aetiology.