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American Journal of Medical Genetics Part C (Seminars in Medical Genetics) ARTICLE

Congenital Microcephaly

DIANA ALCANTARA AND MARK O'DRISCOLL*

The underlying etiologies of genetic congenital microcephaly are complex and multifactorial. Recently, with the exponential growth in the identification and characterization of novel genetic causes of congenital microcephaly, there has been a consolidation and emergence of certain themes concerning underlying pathomechanisms. These include abnormal mitotic microtubule spindle structure, numerical and structural abnormalities of the centrosome, altered cilia function, impaired DNA repair, DNA Damage Response signaling and DNA replication, along with attenuated cell cycle checkpoint proficiency. Many of these processes are highly interconnected. Interestingly, a defect in a gene whose encoded protein has a canonical function in one of these processes can often have multiple impacts at the cellular level involving several of these pathways. Here, we overview the key pathomechanistic themes underlying profound congenital microcephaly, and emphasize their interconnected nature. © 2014 Wiley Periodicals, Inc.

KEY WORDS: cell division; mitosis; DNA replication; cilia How to cite this article: Alcantara D, O'Driscoll M. 2014. Congenital microcephaly. Am J Med Genet Part C Semin Med Genet 9999:1–16.

INTRODUCTION mid‐gestation although glial cell division formation of the various cortical layers. and consequent brain volume enlarge- Furthermore, differentiating and devel- Congenital microcephaly, an occipital‐ ment does continue after birth [Spalding oping neurons must migrate to their frontal circumference of equal to or less et al., 2005]. Impaired neurogenesis is defined locations to construct the com- than 2–3 standard deviations below the therefore most obviously reflected clini- plex architecture and laminar layered age‐related population mean, denotes cally as congenital microcephaly. structure of the cortex [Tan and Shi, a fundamental impairment in normal Fundamentally, neurogenesis incor- 2013; Wu et al., 2014] (Fig. 1). brain development [Woods and Parker, porates several stages that are very What spectrum of physiological 2013]. Depending on the underlying susceptible to problems in the efficient deficits underlies congenital microceph- cause, congenital microcephaly can be and effective execution of genome aly? Defects resulting in elevated levels of associated with structural brain malfor- maintenance, DNA replication and apoptosis can deplete neuroprogenitor mations [e.g., gyrification issues, agenesis ultimately cell division. The developing stem and differentiating cells. Defects of corpus callosum, pituitary abnormal- human neuroepithelium must undergo impacting upon efficient DNA replica- ities] or secondary consequences such as a rapid expansion in stem cell numbers tion can limit the capacity of the craniosynostosis [Verloes et al., 2013]. to fuel its own symmetric expansion neuroepithelium to expand under its Congenital microcephaly can have an [Rakic, 1995]. This is essential to strict temporal constraints. Defects in the environmental or genetic etiology [Gil- generate enough capacity to instigate mitotic apparatus (e.g., microtubule more and Walsh, 2013]. Cerebral corti- and maintain asymmetric division for spindles, centrosomes, centrioles) can cal neurons must have developed by neuronal differentiation, enabling the lead to impaired symmetric‐asymmetric

Grant sponsor: Cancer Research UK; Grant sponsor: Medical Research Council (UK); Grant sponsor: Leukaemia Lymphoma Research (UK). The authors declare no conflicts of interest. Mark O'Driscoll, Ph.D is Research Professor of Human Molecular Genetics in the Genome Damage & Stability Centre [GDSC] at the University of Sussex where he leads the Human DNA Damage Response Disorders Group. His research interests include understanding the mechanisms underlying genome stability, cell cycle regulation, and receptor tyrosine kinase‐mediated signal transduction pathways, with a strong emphasis on dissecting the genotype–phenotype relationships underlying the congential human disorders caused by genetic defects in these fundamental processes. Diana Alcantara, M.Sc, Ph.D is currently a postdoctoral researcher within the Human DNA Damage Response Disorders Group at the GDSC. Her research interests include investigating the wider impacts of ATR‐dysfunction upon the DNA Damage Response and metabolic homeostasis; as well as the molecular causes of congenital human developmental disorders. *Correspondance to: Mark O'Driscoll, Genome Damage & Stability Centre, University of Sussex, Brighton, BN19RQ East Sussex, United Kingdom. E‐mail: [email protected] DOI 10.1002/ajmg.c.31397 Article first published online in Wiley Online Library (wileyonlinelibrary.com).

ß 2014 Wiley Periodicals, Inc. 2 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

Figure 1. Cell division and differentiation in the developing cerebral cortex. (A). Neural plate originating cells initially undergo a phase of symmetric self‐renewal and lateral expansion. Then, as development continues they asymmetrically divide to give rise to glial cells and neurons in the cerebral cortex, which ultimately structures into multiple discrete layers following development. Radial glial cells are key neuroprogenitors whose cell body lies in the ventricular zone (VZ) and whose radial fibers span the width of the cerebral cortex. These can differentiate into neurons, as well as intermediate progenitors that migrate to the subventricular zone (SVZ) and can further generate more progenitor cells and neurons by repeated asymmetric cell division. Finally, radial glial cells also can give rise to astrocytes and oligodendrocytes at later stages. Additionally, the radial glial fibers function as scaffolding for migrating cells to move outwards and away from the VZ to their appropriate destination regions. Ultimately the migrating neurons become the pyramidal cells of the cerebral cortex. CP: cortical plate. IZ: intermediate zone. VZ: ventricular zone. OSVZ: outer‐subventricular zone. ISVZ, inner‐subventricular zone. IZ: intermediate zone. (B) Neuroepithelial cell division within the ventricular zone (VZ) is accompanied by nuclear migration between the apical and basal surfaces of the developing cerebral cortex. In the G1 phase of the cell cycle, the cell nucleus ascends from the apical surface towards the basal end of the ventricular zone, where it remains during S‐phase. During G2, the nucleus then returns to the apical surface where mitosis (M) takes place. (C) Proliferative versus neurogenic (differentiating) cell division is dictated by the cleavage plane orientation (dotted red line) of dividing neuroepithelial (NE) cells. Orientation of the mitotic spindle is parallel to the apical surface during symmetric cell division, with the resulting cleavage plan intersecting the apical plasma membrane (PM). A deviation in the orientation of the mitotic spindle results in an asymmetric cell division, with only one of the daughter cells inheriting the apical PM region (pink cell). The correct orientation of the mitotic spindle is therefore crucial for neurogenesis. The number of rounds of symmetric proliferative divisions undergone by NE cells determines the ultimate number of neurons. As neurogenesis progresses, increased asymmetric divisions means that only one daughter cell inherits the apical PM and remains a NE cell, while the other becomes either a basal progenitor, a radial‐glial cell or a neuron (depicted as the orange cell). Additionally, cell cycle length plays a role in proliferative versus differentiating cell divisions. Longer cell cycles have been associated with differentiating divisions, while shortening of G1 phase for example has been linked to an increase in proliferative divisions.

division, inappropriate cell cycle arrest identifiable microcephalic primordial as mechanisms underlying impaired and/or elevated apoptosis. Mutations dwarfisms (MPDs), such as Seckel neurogenesis. in genes encoding key players in each syndrome, microcephalic primordial of these biological processes have dwarfism types I and II and Meier– MITOSIS AND been described in patients exhibit- Gorlin syndrome. Herein, we overview MICROCEPHALY ing profound congenital microcephaly the genetic defects associated with severe (Tables I–III). This can present as the congenital microcephaly and discuss To date, the majority of the genetic most marked clinical feature; primary how they contribute to the general defects identified in PM and MPDs microcephaly (PM), or in association phenomena of limiting cell division involve genes encoding proteins that with pronounced growth restriction; as capacity and elevating levels of stem play fundamental roles in various pro- in the growing family of genetically and/or neuroprogenitor cell death cesses that collectively enable cells to TABLE I. Centrosome and Spindle Microtubule Defects Underlying Severe Congenital Microcephaly

Gene Protein OMIM Clinical presentation Localization and function MCPH1 Microcephalin 251200 PM. Cells exhibit premature Centrosome‐role in DNA repair chromosome condensation and G2‐M dynamics WDR62 WD‐repeat containing 604317 PM Mitotic spindle pole formation‐scaffold (MCPH2) protein 62 for JNK pathway CDK5RAP2/ Cyclin dependent kinase 5 604804 PM Centrosome, spindle and microtubule CEP215 regulatory subunit‐associated organizing function. (MCPH3) protein 2 CASC5 Cancer susceptibility 604321 PM Kinetochore [KNM] component‐ (MCPH4) candidate 5 spindle‐assembly checkpoint ASPM Abnormal spindle‐like, 608716 PM Microtubule associated protein‐spindle (MCPH5) microcephaly organization and orientation associated CENPJ/CPAP Centromeric protein J 608393 PM‐MPD‐Seckel syndrome Centriole biogenesis‐cilia formation (MCPH6) 613676 STIL (MCPH7) SCL/TAL1 interrupting locus 612703 PM Centrosome‐centriole biogenegis CEP135 Centrosomal protein 135kDa 614673 PM Centrosome‐centriole biogenegis (MCPH8) CEP152 Centrosomal protein 614852 PM‐MPD‐Seckel syndrome Centrosome‐centriole biogenesis (MCPH9) of 152 kDa and genome stability 613823 CEP63 Centrosomal protein 614728 Microcephaly with growth Centrosome‐centriole biogenesis of 63 kDa retardation‐Seckel syndrome [mild] NDE1 Nuclear distribution protein 614019 Microlissencephaly Centrosome‐mitotic spindle nudE homolog 1 microhydraencephaly NIN Ninein 614851 MPD‐Seckel syndrome Centrosome function‐microtubule organization PCNT Pericentrin 210720 Microcephalic Osteodysplastic Component of pericentriolar material‐ Primordial Dwarfism (MOPD) II scaffold for signaling molecules? BUB1B BUB1 Mitotic Checkpoint 257300 Growth retardation‐microcephaly‐ Kinetochore kinase‐role in spindle Serine/Threonine cancer‐Mosaic Variegated checkpoint Kinase B1 (BUBR1) Aneuploidy [MVA] CENPE Centromere protein E 117143a MPD Microtubule capture (CENP‐E) and stabilization KIF5C Kinesin family member 5C 615282 Microcephaly and cortical Microtubule motor protein malformations KIF2A Kinesin family member 2A 615411 Severe microcephaly‐cortical Microtubule motor protein malformations‐early‐onset epilepsy KIF11 Kinesin family member 11 152950 Microcephaly with or without Microtubule motor protein‐role chorioretinopathy, lymphoedema in microtubule crosslinking and mental retardation and bipolar spindle formation TUBG1 Tubulin‐gamma complex 615412 complex cortical malformations‐ Structural component of the associated protein 1 microcephaly (not all reported centrosome‐role in microtubule cases) nucleation TUBB2B Tubulin, beta 2B class IIb 610031 Microcephaly‐spastic Microtubule component‐binds tetraparesis‐severe intellectual to GTP disability‐scoliosis TUBA1A Tubulin, alpha 1A 611603 Microcephaly‐severe Microtubule component intellectual disability POC1A Proteome of the centriole 1A 614783 MPD Centriolar protein required for cilia formation

PM, primary microcephaly; MPD, microcephalic primordial dwarfism. aDenotes the gene entry in OMIM. 4 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

TABLE II. Defects in the Origin Recognition Complex Core and Associated Components Underlying Meier–Gorlin Syndrome

Gene Protein OMIM Clinical presentation Localization & function ORC1 Origin recognition 224690 Meier–Gorlin syndrome Component of the pre‐replication complex subunit 1 complex‐initiation of DNA replication ORC4 Origin recognition 613800 Meier–Gorlin syndrome Component of the pre‐replication complex subunit 4 complex‐initiation of DNA replication ORC6 Origin recognition 613803 Meier–Gorlin syndrome Component of the pre‐replication complex subunit 6 complex‐initiation of DNA replication, coordination of chromosome‐replication and segregation CDT1 Chromatin licensing and 613804 Meier–Gorlin syndrome Component of the pre‐replication complex DNA replication factor 1 ‐origin licensing factor CDC6 Cell division cycle 6 613805 Meier–Gorlin syndrome Component of the pre‐replication complex‐loading MCM complex‐role in cell cycle checkpoints

execute precise chromosomal segrega- an important spindle microtubule orga- with a common clinical manifestation of tion and mitotic division [Thornton and nizing center [Bettencourt‐Dias and congenital microcephaly. Glover, 2007]. There is now an increas- ing list of examples of hypomorphic Centrosomes and Spindles defects in genes encoding core compo- To date, the majority of the nents of the centrosome associated The centrosome cycle is coordinated genetic defects identified in with PM and MPD (Table I and with the canonical cell cycle whereby Figs. 2 and 3). the mother centrosome, inherited from PM and MPDs involve genes Very often, the precise roles of these encoding proteins that play proteins at centrosomes are rather opa- Currently, the working fundamental roles in various que. For some, such as Pericentrin and g‐tubulin, these often have “structural” functional model to explain processes that collectively “ ” or scaffold functions attributed them impaired neurogenesis in the enable cells to execute precise [Zimmerman et al., 2004]. In most context of a defect in a chromosomal segregation instances, descriptive impacts upon cen- triole and centrosome duplication, and centrosomal protein is that and mitotic division. consequently abnormalities in micro- tubule spindle organization, have been these often result in observed for defects in these proteins supernumerary centrosomes, Woods, 2009; Mahmood et al., 2011; [Griffith et al., 2008; Rauch et al., 2008]. Verloes et al., 2013](Table I). The Furthermore, there is growing evidence fragmented centrosomes mitotic phase of the cell cycle involves that defects in some of these proteins and/or premature centriolar an intricate and highly complex ballet of have additional negative impacts upon separation. Consequently, interactions and transactions occurring the centrosomal localization of other in an organized and inter‐dependent centrosome proteins that have indepen- these can result in deficits in fashion [Walczak et al., 2010]. These dently been identified as underlying mitotic spindle microtubule include chromosome condensation, defects of PM and/or MPD. Illustrative bipolar mitotic microtubule spindle examples include the interplay between nucleation when establishing a network formation and dissolution, CEP152 and CEP63 or for CEP152 bipolar spindle. along with chromosomal kinetochore‐ and CENPJ (CPAP) [Cizmecioglu et al., mediated nucleation and capture by 2010; Sir et al., 2011]. These occur- spindle microtubules to instigate the rences further highlight the intercon- the previous mitosis, must duplicate to amphitelic restraining of chromosomes nected and functional interplay between generate a mother‐daughter pair prior for alignment at metaphase prior to many of these proteins, explaining to to G2‐phase where they then act as a segregation. The centrosome represents some degree why defects herein present microtubule organizing center at the TABLE III. Defects in DNA Damage Response and DNA Repair Proteins Associated With Congenital Microcephaly

Gene Protein OMIM Clinical presentation Localization & function ATR Ataxia telangiectasia and 210600 MPD–Seckel syndrome Protein kinase‐apical DDR regulator‐cell cycle rad3‐related checkpoint activation‐role in DNA replication ATRIP ATR‐interacting protein 606605a MPD–Seckel syndrome Essential ATR partner‐binds to RPA‐coated ssDNA‐DDR‐role in DNA replication RBBP8/ Retinoblastoma‐binding 606744 MPD–Seckel syndrome DNA DSB resection‐role in ATR recruitment to CTIP protein 8/CTBP‐interacting DSBs‐associates with BRCA1 in regulation of protein (CtIP) cell cycle checkpoints NBS1/ Nijmegen breakage 251260 Nijmegen breakage syndrome (NBS): Component of M‐R‐N complex with central NBN syndrome 1 /nibrin microcephaly‐immunodeficiency‐ role in DNA DSB repair cancer predisposition RAD50 DNA repair protein RAD50 613078 NBS‐like disorder: microcephaly‐ Component of M‐R‐N complex with central intelletual disability‐“bird‐like” role in DNA DSB repair face‐short stature MRE11A Double‐strand break 600814a NBS‐like severe microcephaly Component of M‐R‐N complex with central repair protein MRE11A role in DNA DSB repair PNKP Bifunctional polynucleotide 613402 Microcephaly‐seizures‐developmental Role in DNA repair pathways (NHEJ, BER) phosphatase and kinase delay CDK6 Cyclin‐dependent kinase 6 603368a PM Control of cell cycle and differentiation‐ centrosomal association in mitosis BRCA1 Breast cancer type 1 113705a Microcephaly‐short stature‐developmental DNA repair‐cell cycle checkpoint control‐ susceptibility protein delay‐cancer predisposition maintenance of genomic stability (HRR) BRCA2 Breast cancer type 2 600185a MPD DNA repair‐cell cycle checkpoint control‐ susceptibility protein maintenance of genomic stability (HRR) LIG4 DNA ligase 4 606593 Ligase IV syndrome: microcephaly‐ DNA DSB repair (NHEJ) and V(D)J dysmorphic facial features‐growth recombination retardation‐skin anomalies‐ pancytopenia. NHEJ1 Non‐homologous 611291 Growth retardation‐microcephaly‐ DNA DSB repair (NHE)] and V(D)J end‐joining factor 1 immunodeficiency recombination CHLR1/ DEAD/H (Asp‐Glu‐Ala‐ 601150a Warsaw breakage syndrome [WABS]: DNA helicase‐genome stability DDX11 Asp/Hi) microcephaly‐pre‐ and postnatal box helicase 11 growth retardation‐abnormal skin pigmentation PHC1 Polyhomeotic‐like protein 1 615414 PM‐short stature Component of polycomb group (PcG) (MCPH11) multiprotein PRC1‐like complex/ repression of transcription/role in chromatin remodelling and histone modification DNA2 DNA replication helicase 2 601810a MPD Helicase‐DNA repair‐genome stability XRCC2 X‐ray repair complementing 600375a Microcephaly‐growth deficiency‐facial DNA DSB repair (HR)‐genome stability defective repair in Chinese nerve palsy‐skeletal abnormalities hamster cells 2 XRCC4 X‐ray repair complementing 194363a MPD DNA DSB repair (NHEJ)‐genome stability defective repair in Chinese hamster cells 4 NHEJ1 Nonhomologous end‐joining 611291 Severe combined immunodeficiency DNA DSB repair [NHEJ]‐genome stability factor 1. (SCID)‐microcephaly‐growth Also called XLF/ retardation‐IR sensitivity Cernnunos RECQL3 Bloom syndrome helicase 210900 Bloom syndrome‐microcephlay‐growth HRR pathway HJ resolving helicase (BLM) delay‐immune deficiency‐cancer

PM, primary microcephaly; IR, ionizing radiation. aDenotes the gene entry in OMIM. MPD: microcephalic primordial dwarfism. HRR: homologous recombination repair. NHEJ: non‐ homologous DNA end joining. M‐R‐N: MRE11‐RAD50‐NBS1 complex. 6 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

Figure 2. Centrioles, centrosome and mitotic microtubule organization. (A) The centriole cycle is intimately coordinated with the canonical cell cycle where centriolar division occurs during S‐phase to ensure mature centrioles are available prior to the onset of mitosis. (B) A normal mitosis is depicted in the upper panel where the centrosomes are shown in black, the chromosomes in gray and the microtubule spindles as the dotted lines. Bi‐polar spindle formation is an essential prerequisite to effective chromosome segregation during mitotic division. The lower panels show mitotic LCLs from a wild‐type (WT) and PCNT‐mutated individual. The spindles were detected using a‐tubulin (green) whilst the centrosomes were stained using g‐tubulin (yellow‐orange). The WT mitotic cell shows a bipolar spindle in contrast to the multipolar spindle from the PCNT‐individual. (Images courtesy of Dr. Iga Abramowicz).

Figure 3. The distribution of genetic defects underlying congenital microcephaly attributable to the centrosome, spindle and kinetochore. The upper panel depicts a normal bipolar mitosis. For the centrosome panel, the proteins in black are centrosome‐specific proteins whilst those in blue are spindle constituent and spindle‐associated proteins associated with congenital microcephaly. For the kinetochore panel CENP‐E is shown extending from the kinetochore via its large coiled‐coiled region onto microtubules. The orange section of CENP‐E denotes its kinesin motor domain. CCAN: constitutive centromere‐associated network. KNM network: KNL1‐ NDC80 (CASC5)‐MIND. ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 7 onset of the next mitosis [Hinchcliffe, congenital microcephaly [Morris‐Rose- congenital microcephaly disorders, as 2001] (Fig. 2A). Currently, the working ndahl et al., 2008; Jaglin and Chelly, PM and/or MPD. functional model to explain impaired 2009; Romaniello et al., 2014] (Table I neurogenesis in the context of a defect in and Fig. 3). These defects are typically a centrosomal protein is that these often also associated with marked deficits in DNA REPLICATION, CILIA result in supernumerary centrosomes, cortical development (e.g., lissencephaly, FUNCTION AND fragmented centrosomes and/or prema- pachygyria, polymicrogyria) due to MICROCEPHALY ture centriolar separation [Woods and abnormalities in neuronal migration Parker, 2013]. Consequently, these can (e.g., KIF2A, KIF5C) [Poirier et al., Compared to other cell types, cell cycle result in deficits in mitotic spindle 2013]. length can be remarkably short in microtubule nucleation when establish- developing neuroprogenitors [Rakic, ing a bipolar spindle. Very often, 1995]. Since these cells need to undergo The Kinetochore and Spindles multipolar spindles are observed in rapid and temporally restricted expan- patient‐derived cell lines representing a Considering its role in microtubule sion, efficient DNA replication is fun- catastrophic consequence for mitosis, capture and chromosome segregation, damental to ensure normal neuronal chromosome segregation and cell divi- defects in kinetochore components as an development. In fact, even within sion [Rauch et al., 2008; Issa et al., 2013] underlying pathomechanism for con- progenitor populations there appears to (Fig. 2B). Such outcomes can result in genital microcephaly are very much be significant variation in the duration of permanent cell cycle arrest via activation under‐represented to date, compared certain cell cycle phases; most notably ‐ of the spindle assembly checkpoint to the spindle and centrosome (Table I G1 and S phase, depending upon the (SAC), as well as cytokinesis failure and Fig. 3). Mutations in BUB1B cause specific lineage commitment of the and subsequent apoptosis [Musacchio, mosaic variegated aneuploidy, an MPD progenitors in question [Dehay et al., 2011]. All of these impacts could limit associated with elevated cancer predis- 2001; Dehay and Kennedy, 2007; Pilaz neuroepithelial stem cell maintenance position, particularly Wilms tumor et al., 2009; Arai et al., 2011]. Therefore, and expansion, as well as disrupt the [Matsuura et al., 2000; Hanks et al., genetic defects that can adversely impact important balance between cell division 2004]. BUB1B encodes BUBR1, a SAC upon the duration of these cell cycle and differentiation (Fig. 1). protein that localizes to the kinetochore phases could potentially have a dramatic Defects in microtubule spindle of lagging chromosomes during mitosis effect upon the efficiency of cortical components and spindle‐associated pro- [Bolanos‐Garcia et al., 2009; Kiyomitsu development. teins represent the most frequent under- et al., 2011]. SAC activation ensures all lying cause of congenital microcephaly kinetochores have robust amphitelic described to date (Table I). One well microtubule attachments prior to segre- Origin Licensing, G1‐S Transition, known example is ASPM (abnormal gation [Rudner and Murray, 1996; DNA Replication and S‐Phase spindle‐like microcephaly‐associated Musacchio, 2011]. Progression protein), a spindle binding protein that A defect in CASC5 was reported Recently, defects in multiple compo- localizes to the pericentriolar matrix in three related families used to define nents of the origin recognition complex (PCM) of the centrosome at the onset of the original MCPH4 locus [Jamieson mitosis [Bond et al., 2002]. It has been et al., 1999; Genin et al., 2012]. CASC5 shown that defects in ASPM function encodes a component of the KMN Recently, defects in multiple can result in altered spindle pole orien- Complex [KNL1‐Mis12 complex‐ tation in the developing neuroepithe- Ndc80], a kinetochore localizing components of the origin lium, thereby disrupting the balance multi‐protein complex involved in mi- recognition complex [ORC], between symmetric and asymmetric crotubule stabilization and SAC silenc- a multi‐subunit complex that division of neuronal stem cells [Fish ing [Kiyomitsu et al., 2007]. Mirzaa et al. et al., 2006] (Fig. 1C). Indeed this has [2014] recently described the first ex- ‘licenses’ and thereby initiates also recently been elegantly demonstrat- ample of a defect in a core kinetochore DNA replication from mainly ed in the developing brain of a mouse component as the underlying cause of ‐ model of Mcph1 (Microcephalin), a MPD. They found defects in CENPE, non sequence specific discrete common cause of PM in humans the gene encoding the large kinetochore genomic regions, were [Gruber et al., 2011]. Defects in micro- protein CENP‐E which plays a vital role ‐ tubule and cytoskeletal constituents in microtubule capture during mitosis identified in Meier Gorlin (e.g., a‐ and b‐tubulin) and even (Fig. 3). Cells from the affected individ- syndrome (MGS). microtubule interacting proteins that uals exhibited multiple interconnected regulate diverse processes such as micro- mitotic abnormalities. It is likely that tubule formation, stabilization and de- other kinetochore‐associated defects [ORC], a multi‐subunit complex that polymerization, can also result in await to be identified as novel causes of “licenses” and thereby initiates DNA 8 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE replication from mainly non‐sequence Cilia‐Function and S‐Phase Entry implicated in multiple post‐mitotic specific discrete genomic regions, were neuronal functions, suggestive of addi- identified in Meier‐Gorlin syndrome With respect to the impaired origin tional roles outside of DNA replication (MGS) [Bicknell et al., 2011a,b; Guern- licensing and delayed S‐phase kinetics origin licensing (reviewed in Kerzen- sey et al., 2011] (Table II). MGS is an observed in MGS, subsequent investiga- dorfer et al., 2013). MPD associated with additional features tion provided evidence to suggest a more Is there precedence for centrosome‐ including endochondral ossification ab- complex and multifaceted pathome- based cilia dysfunction in human con- normalities [Gorlin et al., 1975; Ahmad chanism. Stiff et al. [2013] found that genital microcephaly disorders? Defects and Teebi, 1997; Bongers et al., 2005]. while MGS‐causative defects were asso- in PCNT, encoding the centrosomal With respect to ORC1‐mutated MGS, ciated with reduced licensing of an protein Pericentrin, underlie the MPD specific deficiencies in DNA origin ectopically supplied DNA replication microcephalic primordial osteodysplas- licensing, DNA replication initiation, origin, unexpectedly, some of these tic dwarfism type‐II (MOPD‐II) [Grif- G1‐S transition and S‐phase progression defects did not segregate with delayed fith et al., 2008; Rauch et al., 2008]. were catalogued in patient lymphoblas- S‐phase progression in patient LCLs. Impaired PCNT function is associated toid cell lines (LCLs), thus suggesting a Rather, all of these defects were associ- with cilia‐dysfunction [Miyoshi et al., pathomechanism based upon delayed ated with centriole‐centrosome abnor- 2006, 2009; Mühlhans et al., 2011]. DNA replication as underlying the malities, impaired cilia formation and Multiple mutations in POC1A which clinical presentation [Bicknell et al., consequently cilia‐dependent signaling encodes a centriole protein, have been 2011b]. Furthermore, several ORC1‐ [Stiff et al., 2013]. Interestingly, ORC1 identified in several MPD families MGS mutations localized to the BAH can localize to the centrosome in a [Shaheen et al., 2012]. Interestingly (bromo adjacent homology) domain of Cyclin A‐dependent manner and has these defects were associated with ORC1 [Bicknell et al., 2011b]. This previously been implicated in control- centrosome fragmentation, microtubule region was shown to bind histone H4‐ ling centriole and centrosome copy spindle abnormalities (e.g., multipolar lysine‐20‐dimethylated (H4‐K12‐Me2), number via interaction with Cyclin E spindles) as well as cilia formation and and since H4‐K12‐Me2 is enriched at [Hemerly et al., 2009]. Whether other signaling deficits [Shaheen et al., 2012]. two known human replication origins, ORC components have direct roles at In fact, ASPM has recently been it has been postulated that this domain the centrosome and/or cilia is not clear. associated with compromised WNT is required for ORC‐recruitment to Cilia formation and function are signaling which could also reflect an origins [Noguchi et al., 2006; Kuo vital for coordinating cell cycle entry underlying problem in cilia function et al., 2012]. Collectively, these findings from G0 phase into G1‐S [Heldin and [Ponting, 2006; Buchman et al., 2011]. appear to support a model whereby a Westermark, 1999; Schneider et al., Therefore, it would appear that direct impact upon DNA replication 2005]. Furthermore, cilia function is clinically relevant cilia abnormalities kinetics, due to defects in components intimately associated with neuronal impacting on neurogenesis can originate that initiate DNA replication, are asso- development [Spassky et al., 2008; Han from at least two routes; firstly, as a direct ciated with congenital microcephaly in and Alvarez‐Buylla, 2010; Lee and consequence of defects in proteins with the context of this MPD. Gleeson, 2011]. Delayed G1‐S transit known and/or probable roles in cilia Recent findings suggest that there is appears to be a feature of MGS cells and formation which emanates from the more to this basic model. For example, this delay appears also to be dependent centriolar‐basal body, and secondly, pathogenic defects in MCM4, a core upon cilia‐signaling [Stiff et al., 2013]. from the likely secondary or indirect component of the replisome, have This is an intriguing result considering consequence of defects in proteins been identified in patients with adrenal the importance of G1 phase length in without prescribed roles in the cilium insufficiency, growth restriction and a regulating the balance between neuro- or in cilia formation, as in the case of the selective Natural Killer cell defect, progenitor stem cell regeneration and origin licensing components. The latter although without any overt indication progenitor lineage commitment via route may prove to be more widespread of congenital microcephaly [Casey et al., differentiation [Dehay and Kennedy, than currently appreciated. 2012; Gineau et al., 2012; Hughes 2007; Pilaz et al., 2009]. The cilia‐ et al., 2012]. Similarly, a polymerase‐ dependent findings in MGS originating THE DNA DAMAGE inactivating defect in the catalytic sub- from defects in components with ca- RESPONSE (DDR) AND unit (POLD1) of DNA polymerase d, nonical roles in DNA replication adds an MICROCEPHALY the lagging strand DNA polymerase, extra layer of complexity to our under- has been identified in an individual with standing of the molecular and cellular The PI3‐kinase‐like family members a complex disorder involving lipodys- impacts that converge here to adversely ATM (Ataxia Telangiectasia Mutated) trophy, deafness, hypogonadism and affect normal neuronal development and ATR (Ataxia Telangiectasia and mandibular hypoplasia; again, without (and height attainment and endochon- Rad3‐related) are the apical protein overt congenital microcephaly [Weedon dral ossification). Additionally, several kinases of the DNA Damage Response et al., 2013]. ORC components have also been (DDR) [Cimprich and Cortez, 2008; ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 9

Lavin, 2008; Maréchal and Zou, 2013; microcephaly extending to 9to12 neurogenesis, and then only in certain Shiloh and Ziv, 2013]. The DDR is a SD below the age related mean post‐ progenitor populations [Lee et al., signal transduction cascade activated natally [Ogi et al., 2012]. Some 2012b]. Rapid proliferation of granule upon detection of DNA strand breaks problems in myelination and isolated neurons within the embryonic cerebellar that initiates and controls integrated structural abnormalities (e.g., absent external germ layer (EGL) occurs in responses to these damages [Sirbu and pituitary fossa, agenesis of corpus response to sonic hedgehog (Shh), Cortez, 2013]. These responses include callosum) have also been observed in whose mitogenic potential is realized cell cycle checkpoint activation, DNA these patients, although not consistently via cilia [Spassky et al., 2008]. Atr‐ replication fork stabilization, engage- (Fig. 4A) [Ogi et al., 2012]. deficient EGL cells underwent p53‐ ment of DNA repair pathways and ATR plays a vital role in stabilising independent proliferation arrest, while ultimately apoptosis, if the extent of stalled DNA replication forks [Cimprich other areas underwent p53‐dependent damage precludes survival. ATM is and Cortez, 2008]. Both ATM and ATR apoptosis [Lee et al., 2012b]. Further- activated by DNA double strand break- function in controlling dormant DNA more, co‐incident inactivation of Atm age (DSB) while ATR is activated by replication origin firing and activation was unexpectedly found not to exacer- RPA‐coated single stranded DNA of G1‐S, intra‐S and G2‐M cell cycle bate Atr loss in the brain, suggesting a (ssDNA) which can occur upon stalling checkpoints. One of the first bona fide non‐overlapping role for each kinase of DNA replication forks or as an substrates identified and characterized in this developmental context [Lee intermediate during certain DNA for these kinases was p53 [Banin et al., 2012b]. repair processes (e.g., DSB resection et al., 1998]. There are now many others Topoisomerase II binding protein I to facilitate homologous recombination [Matsuoka et al., 2007; Stokes et al., (TopBP1) is essential for DNA replica- repair). Both kinases largely function 2007]. Therefore, both kinases directly tion and cell cycle checkpoint activation in a redundant manner in the DDR signal to the apoptotic machinery via this [Sokka et al., 2010]. It also plays a key [O’Driscoll and Jeggo, 2008]. and other routes [Roos and Kaina, role in activating ATR kinase [Kumagai Congenital deficiency of ATM 2006]. Loss of ATR function is associat- et al., 2006]. Interestingly, conditional results in ataxia telangiectasia [A–T], a ed with elevated replication fork collapse progenitor‐restricted deletion of Topbp1 cerebellar neurodegenerative condition and consequently DSB formation, was found to be essential for early involving the progressive loss of Purkinje therefore triggering an ATM‐dependent progenitor genome stability and survival, neurons specifically [Lavin, 2008]. DDR cascade. This was elegantly dem- but not, unexpectedly, for replication per Hypomorphic defects in ATR underlie onstrated as a physiological consequence se [Lee et al., 2012a]. Furthermore, this Seckel syndrome; the archetypal MPD of hypomorphic ATR‐ablation in a impact was found to be p53‐dependent mouse model of ATR‐mutated Seckel whilst Atm‐independent [Lee et al., syndrome (AtrS/S) [Murga et al., 2009]. 2012a]. Atm has previously been shown Embryos of the AtrS/S animal exhibited to be an important mediator of p53‐ Hypomorphic defects in ATR massively elevated spontaneous levels of dependent DSB‐induced apoptosis in underlie Seckel syndrome; the replicative stress‐induced DNA damage the nervous system [Herzog et al., 1998]. archetypal MPD. ATR has and apoptosis, even in the developing Collectively, these findings highlight the neuroepithelium [Murga et al., 2009]. importance of the cell‐specific context an obligate co‐stabilising The surviving animals exhibited cranio- within the brain, along with the nature binding partner called ATRIP facial abnormalities (receding forehead, of the genomic instability (e.g., replica- ‐ ‐ micrognathia), growth restriction and tion fork dependent), as being funda- (ATR Interacting Protein), congenital microcephaly reminiscent of mental to the precise impacts of defects and defects herein have also the ATR‐mutated Seckel syndrome in DDR‐signaling controllers such as individuals, consistent with the concept Atr, Atm and Topbp1, in the developing been identified as a cause of of intrauterine programming [Murga mouse brain at least. Seckel syndrome. et al., 2009; O’Driscoll, 2009]. DEFECTIVE DNA DOUBLE Defective DDR, Apoptosis and STRAND BREAK [DSB] [O’Driscoll et al., 2003]. ATR has an Microcephaly REPAIR AND obligate co‐stabilizing binding partner MICROCEPHALY called ATRIP (ATR‐interacting pro- Conditional Cre‐restricted mouse mod- tein), and defects herein have also been els have shown that the functional inter‐ Non‐homologous DNA End‐Joining identified as a cause of Seckel syndrome relationships between in the apical DDR (NHEJ) and Homologous Recombina- [Ogi et al., 2012]. From an neuroana- kinases are more complex when consid- tion Repair (HRR) are the core DSB tomical perspective, impaired ATR‐ ering brain development. For example, repair pathways [O’Driscoll and Jeggo, ATRIP function is most overtly conditional Atr ablation was unexpect- 2006; Moynahan and Jasin, 2010; associated with marked congenital edly found to impact relatively late upon Deriano and Roth, 2013]. Interestingly, 10 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

Figure 4. Defective DDR and DNA repair as a cause of congenital microcephaly. (A) Neuroanatomical images of the ATRIP‐mutated Seckel syndrome individual described by [Ogi et al., 2012]. The patient is severely microcephalic [10 SD] with evidence of an abnormally shaped pituitary (without an obvious fossa). (Images courtesy of Dr. Margaret Barrow). (B) Homologous recombination repair (HRR) from a DSB is initiated by exonunclease‐mediated resection of the DSB ends. These RPA‐coated single stranded overhangs are then bound by RAD51 generating a filament structure that can invade the sister chromatid to form a D‐loop. The crossover point is referred to as a Holliday Junction (HJ). Template driven replication occurs and is followed by second end capture to generate a crossover containing a double HJ (dHJ). This intertwined molecule requires resolution which can occur via BLM helicase‐dependent route, which generates a non‐crossover product, or, a nuclease‐dependent route to generate a cross‐over. The nucleases that can act on this structure are termed “resolvases.” (C)An on‐going DNA replication fork is shown with the parental DNA in black and the newly replicated DNA in blue. If this structure collides with a DNA single strand break (SSB) the resultant product can contain a DNA double strand break (DSB). This situation requires the coordinate action of DDR mechanisms and distinct DNA repair pathways. The same situation can also occur if an active or moving transcription fork was to collide with a SSB. (D) Some of the damaged or modified DNA strand ends that can occur following oxidative damage to DNA. The normal end polarity of a 50 phosphate and 30 hydroxyl group must be restored in order to allow enzymatic ligation of a break. TopoI denotes a stabilized TopoisomeraseI cleavable complex (CC) which is a normal intermediate in TopoI’s action on DNA. This enzyme normally introduces a SSB to release torsional tension ahead of on‐going replication and transcription forks. These breaks are normally dealt with by the SSBR machinery.

congenital defects in DSB repair mech- ligation [Deriano and Roth, 2013]. This Non‐Homologous DNA anisms are frequently associated with processing can potentially result in the End‐Joining [NHEJ] congenital microcephaly [O’Driscoll loss of genetic material. HRR on the and Jeggo, 2008]. Mouse studies suggest other‐hand requires the presence of a NHEJ is required for immunoglobin that both pathways are essential for sister chromatid to act as a template for and T cell receptor generation via the developmental viability in mammals; repair via strand invasion from one end V(D)J and Class Switch Recombination an indication that substantial levels of of the DSB during Holliday Junction mechanisms [Gellert, 2002]. Therefore, spontaneous DSBs can be generated (HJ) formation [Heyer et al., 2010] congenital defects in NHEJ pathway during development [Symington and (Fig. 4B). Therefore, this pathway has components are also frequently associat- Gautier, 2011]. NHEJ involves direct re‐ been assumed to only be operational ed with variable immunodeficiency, ligation of DSBs, sometimes necessitat- during cell cycle phases where a sister ranging from variable/combined immu- ing processing of damaged base and/or chromatid is present (i.e., late S‐G2‐M) nodeficiency to severe combined immu- sugar moieties at DSB termini prior to [Symington and Gautier, 2011]. nodeficiency [O’Driscoll and Jeggo, ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 11

2006]. Todate, human defects have been erozygous) in the HRR genes RAD51C nuclease implicated in Okazaki fragment identified in PRKCD encoding DNA‐ and RAD51D have been identified in processing and the processing of abnor- PKcs, DCLRE1C encoding ARTEMIS, breast and ovarian cancer cohorts mally structured replication forks (e.g., LIG4 encoding DNA ligase IV and the [Meindl et al., 2010; Loveday et al., aberrant recombination intermediates) genes encoding XRCC4 and XLF/ 2011, 2012; Shaheen et al., 2014]. This [Mimitou and Symington, 2009; Kang Cernunnos [Moshous et al., 2001; suggesting that HRR defects can have a et al., 2010]. A truncating DNA2 O’Driscoll et al., 2001; Buck et al., highly variable clinical presentation. mutation has recently been identified 2006; van der Burg et al., 2009; Wood- in a consanguineous MPD family bine et al., 2013; Murray et al., 2014; Helicases and Nucleases [Shaheen et al., 2014]. This category Shaheen et al., 2014]. Interestingly, of defect reinforces the concept that an ARTEMIS‐defective patients, uniquely The Bloom syndrome helicase BLM impaired ability to process DNA breaks in this context, do not appear to exhibit [RECQL3] plays an important role in HJ during DNA replication may represent microcephaly, in contrast to the other resolution, and congenital microcephaly a contributing pathomechanism to con- genes [Moshous et al., 2001]. This may can be marked in this condition genital microcephaly. be a consequence of the magnitude and [Hickson, 2003; Croteau et al., 2014] Prior to RAD51‐mediated strand nature of their DSB repair; ARTEMIS‐ (Fig. 4B). Warsaw breakage syndrome, a invasion to establish HJs during HRR, disorder of severe congenital microceph- defective cells exhibit a specific DSB DSBs in S‐G2 undergo resection repair in heterochromatin; an ATM‐ aly, growth retardation and chromosom- (Fig. 4B). Various exonucleases have dependent process [Riballo et al., 2004]. al instability collectively reminiscent of been implicated in this process, one FA and the cohensinopathies, was found of best known being the MRE11‐ to result from a mutation in another RAD50‐NBN (M‐R‐N) complex [Sy- Homologous Recombination ‐ ATP dependent DNA helicase, mington and Gautier, 2011]. ATM Repair CHLR1 (DDX11) [van der Lelij et al., directly phosphorylates each component By contrast to NHEJ, congenital defects of this complex following DSB forma- in core components of the HRR tion. Mutations in all M‐R‐N compo- machinery are still relatively rare in The Bloom syndrome helicase nents have now been identified in humans, although some isolated germ‐ clinically overlapping human disorders line defects have been described [re- BLM [RECQL3] plays an exhibiting congenital microcephaly. De- viewed in O’Driscoll, 2012]. Recent important role in HJ fects in NBN encoding Nibrin/NBS1 examples include XRCC2 in a patient cause Nijmegen breakage syndrome exhibiting fanconi anemia (FA), resolution, and congenital (NBS), a disorder of congenital micro- BRCA1 in a woman with early onset microcephaly can be marked in cephaly, growth restriction and haema- ovarian cancer and microcephaly, and this condition. Warsaw tological malignancy [Carney et al., BRCA2 in the context of MPD [Sham- 1998; Varon et al., 1998]. Defects in seldin et al., 2012; Domchek et al., 2013; breakage syndrome, a disorder RAD50 have been shown to cause an Shaheen et al., 2014]. FA patients of severe congenital NBS‐like disorder, whilst mutations in frequently exhibit congenital micro- MRE11 are associated with a clinical cephaly although bone marrow failure microcephaly, growth spectrum ranging from A‐T‐Like Dis- and acute myeloid leukaemia develop- retardation and chromosomal order (A‐T‐LD) without overt micro- ‐ ment are the typically invariant features instability collectively cephaly to an NBS like condition, of this condition [Kee and Andrea, probably reflective of different geno- 2012]. The FA pathway is functionally reminiscent of FA and the type‐phenotype impacts [Stewart et al., integrated with HRR and defects in cohensinopathies, was found 1999; Waltes et al., 2009; Matsumoto other core HRR components have et al., 2011]. CtIP (CTBP‐interacting emerged clinically as FA; the classical to result from a mutation in protein) is another of these exonucleases example being BRCA2 mutations in another ATP‐dependent implicated in DSB resection [Mimitou FA individuals of the FANC‐D1 com- and Symington, 2009]. CtIP is encoded plementation group [Moldovan and DNA helicase, CHLR1 by RBBP8 (retinoblastoma binding pro- D’Andrea, 2009; Kim and D’Andrea, (DDX11). tein 8), wherein a hypomorphic muta- 2012]. Other examples include tion has been described in a Seckel RAD51C (FANC‐O), PALB2, encod- syndrome kindred [Qvist et al., 2011]. ing the BRCA2 interacting protein 2010; Capo‐Chichi et al., 2013]. This Therefore, hypomorphic defects in (FANC‐N) and SLX4 (FANC‐P), en- helicase has been implicated in chromo- various exonucleases that function dur- coding a component of the SLX4‐SLK1 some cohesion and in the recovery ing specific stages of DSB repair are HJ resolving endonuclease (Fig. 4B). of replicating cells from DNA damage emerging as an underlying cause of Interestingly, germ‐line mutations (het- [Shah et al., 2013]. DNA2 is a helicase‐ congenital microcephaly. 12 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

DSB Repair Pathways in the normal neurogenesis often resulting phosphatase (PNKP). Multiple novel Developing Brain: Spatial in congenital microcephaly [O’Driscoll mutations in PNKP were recently Regulation and Checkpoint described in several individuals exhibit- Activation ing severe primary microcephaly, devel- opmental delay, hyperactivity and As mentioned above, HRR is by its seizures (MCSZ) [Shen et al., 2010]. nature of requiring a homologous DNA A growing body of evidence Interestingly, PNKP’s dual kinase and strand as a template for repair, restricted now indicates that the phosphatase activity, which is essential to specific phases of the cell cycle where inability to repair DSBs for repair of the aberrantly modified sister chromatids are available (i.e., S‐G ‐ 2 termini at strand breaks, is also implicat- M) (Fig. 4B). Therefore, tissues with a because of defects in NHEJ ed in NHEJ (Fig. 4D) [Caldecott, 2002; very high replicative index would be and/or HRR clearly adversely Koch et al., 2004; Weinfeld et al., 2011]. expected to rely heavily on this pathway Therefore, the marked congenital mi- to repair DSBs, compared to non‐ impacts upon normal crocephaly in MCSZ may be more replicative post‐mitotic tissues. Studies neurogenesis often resulting in reflective of combined attenuation of using knockout embryonic mouse mod- congenital microcephaly. both SSBR and DSB repair pathways. els of NHEJ and HRR have suggested that both these DSB repair pathways may have distinct spatiotemporal func- and Jeggo, 2008; O’Driscoll, 2012]. CONCLUDING REMARKS tionality during neuronal development There has been much speculation as to Congenital microcephaly has a complex [Orii et al., 2006]. Defective HRR what precisely could be the origin of underlying genetic basis, as demonstrat- (Xrcc2)‐induced apoptosis appeared to endogenous DSBs during neurogenesis. ed by the selection of defects discussed occur predominantly within the prolif- One suspect, because of the high oxygen here. An inability to divide and differ- erating neuronal precursors of the consumption and metabolic rate of entiate effectively resides at the center of ventricular zone (VZ) [Orii et al., neurons and their supportive cells, is this and there are multiple routes 2006] (Fig. 1A). In contrast, defective endogenously generated reactive oxy- through which these processes can be NHEJ (Lig4)‐induced apoptosis appear- gen species (ROS)‐induced oxidative disrupted. The growing number of ed restricted to the postmitotic differen- DNA damage [Caldecott, 2008]. This causative genetic defects described for tiating neurons of the subventricular can result in base and ribose sugar congenital microcephaly consolidates zone (SVZ) [Orii et al., 2006]. Work backbone damage/modifications, abasic the importance of mitotic spindle using ionising radiation (IR)‐induced apurinic/apyrimidinic (AP) site forma- organization and centrosome stability DSBs in a hypomorphic Lig4 embryonic tion (i.e., base loss) and even overt single in enabling the execution of precise mouse model (Lig4Y288C) has further strand break (SSB) formation. These chromosome segregation during mito- developed this concept by providing lesions are rapidly dealt with by the sis. Novel causative genetic defects also evidence for a functional, but relatively coordinated action of the base excision provide evidence that perturbations of insensitive, G2‐M cell cycle checkpoint repair (BER) and single strand break other cell cycle phases are also relevant to in the VZ‐SVZ [Gatz et al., 2011]. This repair (SSBR) pathways [Caldecott, normal neurogenesis, for example, insensitive checkpoint allows cells with 2008]. If an active DNA replication or DNA replication origin licensing, G1‐ low numbers of DSBs to transit from the transcription fork encounters an SSB S entry, DNA synthesis and efficient S‐ VZ‐SVZ to the intermediate zone (IZ)‐ there is an elevated risk of consequent phase progression. The importance and cortical plate (CP) region where they DSB formation (Fig. 4C). integration of functional cilia‐signaling then die by apoptosis [Gatz et al., 2011]. Congenital defects in certain SSBR in the context of regulated cell cycle Interestingly, work in somatic cells has components have been identified in entry and progression cannot be over- shown that the DSB‐induced G2‐M humans, but these are usually associated stated. Finally, defects in multiple com- cell cycle checkpoint can be quite an with slowly progressive cerebellar de- ponents of the complex pathways that ineffective block to preventing cells with generation, ultimately presenting with control genome stability and integrity modest levels of DSBs from entering ataxia and peripheral neuropathy‐disor- via signal transduction and repair pro- mitosis [Lobrich and Jeggo, 2007]. ders, rather than congenital microceph- cesses are additional important contrib- aly [Caldecott, 2008; O’Driscoll, 2012]. utors to congenital microcephaly. It is likely that active and complementary Without doubt, the advent of Origins of Endogenous DNA DNA‐repair pathways co‐ordinately exome sequencing has greatly facilitated Breaks in the Developing Brain play a protective role during neuro- the identification of novel microcepha- A growing body of evidence now genesis in this context. One example of ly‐causing defects, further developing indicates that the inability to repair a defect in a core SSBR component our understanding of the molecular basis DSBs because of defects in NHEJ and/ where this complementarity may be of this abnormality. This trend is likely to or HRR clearly adversely impacts upon compromised is polynucleotide kinase‐ continue. The consequent challenge in ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 13 future will be functionally validating and Bettencourt‐Dias M, Glover DM. 2007. Centro- Casey JP,Nobbs M, McGettigan P,Lynch S, Ennis some biogenesis and function: Centrosomics S. 2012. Recessive mutations in MCM4/ determining the pathogenicity of the brings new understanding. Nat Rev Mol PRKDC cause a novel syndrome involving a multitude of candidate variants derived Cell Biol 8:451–463. primary immunodeficiency and a disorder of from such approaches. This issue is Bicknell LS, Bongers EMHF, Leitch A, Brown S, DNA repair. J Med Genet 49:242–245. Schoots J, Harley ME, Aftimos S, Al‐Aama Cimprich KA, Cortez D. 2008. ATR: An essential compounded by the multigenic occur- JY, Bober M, Brown PAJ, van Bokhoven H, regulator of genome integrity. Nat Rev Mol rence of variants, which is still likely an Dean J, Edrees AY, Feingold M, Fryer A, Cell Biol 9:616–627. under‐appreciated and under‐reported Hoefsloot LH, Kau N, Knoers NVAM, Cizmecioglu O, Arnold M, Bahtz R, Settele F, MacKenzie J, Opitz JM, Sarda P, Ross A, Ehret L, Haselmann‐Weiß U, Antony C, situation at present [Agha et al., 2014]. TempleIK, Toutain A, Wise CA, Wright M, Hoffmann I. 2010. Cep152 acts as a scaffold Functional cellular biology using pa- Jackson AP. 2011a. Mutations in the pre‐ for recruitment of Plk4 and CPAP to the tient‐derived cell lines, model cell replication complex cause Meier–Gorlin centrosome. J Cell Biol 191:731–739. syndrome. Nat Genet 43:356–359. Croteau DL, Popuri V, Opresko PL, Bohr VA. systems (siRNA, shRNA, cDNA com- Bicknell LS, Walker S, Klingseisen A, Stiff T,Leitch 2014. Human RecQ helicases in DNA plementation strategies) and even iPS A, Kerzendorfer C, Martin C‐A, Yeyati P,Al repair, recombination, and replication. from patients’ cells, will likely continue Sanna N, Bober M, Johnson D, Wise C, Annu Rev Biochem 83.Epub ahead of print. Jackson AP, O’Driscoll M, Jeggo PA. 2011b. PMID 24606147. DOI: 10.1146/annurev‐ to remain the basic cornerstone ap- Mutations in ORC1, encoding the largest biochem‐060713‐035428 proaches in helping to meet this chal- subunit of the origin recognition complex, Dehay C, Kennedy H. 2007. Cell‐cycle control lenge. The growing use of more cost‐ cause microcephalic primordial dwarfism and cortical development. Nat Rev Neurosci resembling Meier–Gorlin syndrome. Nat 8:438–450. effective and rapidly growing model Genet 43:350–355. Dehay C, Savatier P, Cortay V, Kennedy H. 2001. organisms in this area, such as zebrafish, is Bolanos‐Garcia VM, Kiyomitsu T, D’Arcy S, Cell‐cycle kinetics of neocortical precursors also likely to continue [Novorol et al., Chirgadze DY, Grossmann JG, Matak‐ are influenced by embryonic thalamic axons. Vinkovic D, Venkitaraman AR, Yanagida J Neurosci 21:201–214. 2013]. For now, mouse models remain M, Robinson CV, Blundell TL. 2009. The Deriano L, Roth DB. 2013. Modernizing the effective ‘gold standard’ for neuro- crystal structure of the N‐terminal region of the nonhomologous end‐joining repertoire: genic‐microcephaly research, but the BUB1 provides insight into the mechanism alternative and classical NHEJ share the stage. of BUB1 recruitment to kinetochores. Annu Rev Genet 47:433–455. potential offered by the fascinating Structure 17:105–116. Domchek SM, Tang J, Stopfer J, Lilli DR, Hamel cerebral organoid system represents a Bond JRE, Mochida GH, Hampshire DJ, Scott S, N, Tischkowitz M, Monteiro ANA, Messick very exciting future prospect [Lancaster Askham JM, Springell K, Mahadevan M, TE, Powers J, Yonker A, Couch FJ, Goldgar Crow YJ, Markham AF, Walsh CA, Woods DE, Davidson HR, Nathanson KL, Foulkes et al., 2013]. CG. 2002. ASPM is a major determinant WD, Greenberg RA. 2013. Biallelic delete- of cerebral cortical size. Nat Genet 32:316– rious BRCA1 mutations in a woman with 320. early‐onset ovarian cancer. Cancer Discov ACKNOWLEDGMENTS Bongers E, Van Kampen A, Van Bokhoven H, 3:399–405. Knoers N. 2005. Human syndromes with Fish JL, Kosodo Y,Enard W,Pääbo S, Huttner WB. Work in the O’Driscoll laboratory is congenital patellar anomalies and the under- 2006. Aspm specifically maintains symmetric supported by program funding from lying gene defects. Clin Genet 68:302–319. proliferative divisions of neuroepithelial Buchman JJ, Durak O, Tsai L‐H. 2011. ASPM cells. Proc Natl Acad Sci USA 103:10438– Cancer Research UK with additional regulates Wnt signaling pathway activity in 10443. support from the Medical Research the developing brain. Genes & Development Gatz SA, Ju L, Gruber R, Hoffmann E, Carr AM, Council (UK) and Leukaemia Lympho- 25:1909–1914. Wang Z‐Q, Liu C, Jeggo PA. 2011. Buck D, Malivert L, de Chasseval R, Barraud A, Requirement for DNA Ligase IV during ma Research (UK). Thanks to Iga Fondaneche M‐C, Sanal O, Plebani A, Embryonic Neuronal Development. J Neu- Abramowicz and Margaret Barrow for Stephan J‐L, Hufnagel M, le Deist F, Fischer rosci 31:10088–10100. images. A, Durandy A, de Villartay J‐P,Revy P.2006. Gellert M. 2002. V[D]J recombination: RAG Cernunnos, a novel nonhomologous end‐ proteins, repair factors, and regulation. Annu joining factor, is mutated in human immu- Rev Biochem 71:101–132. nodeficiency with microcephaly. Cell 124: Genin A, Desir J, Lambert N, Biervliet M, Van Der REFERENCES 287–299. Aa N, Pierquin G, Killian A, Tosi M, Urbina Caldecott K. 2002. Polynucleotide kinase. A M, Lefort A, Libert F, Pirson I, Abramowicz Agha Z, Iqbal Z, Azam M, Siddique M, Willemsen versatile molecule makes a clean break. M. 2012. Kinetochore KMN network gene MH, Kleefstra T, Zweier C, de Leeuw Structure [Camb] 10:1151. CASC5 mutated in primary microcephaly. N, Qamar R, van Bokhoven H. 2014. A Caldecott KW. 2008. Single‐strand break repair Hum Mol Genet 21:5306–5317. complex microcephaly syndrome in a Pak- and genetic disease. Nat Rev Genet 9:619– Gilmore EC, Walsh CA. 2013. Genetic causes of istani family associated with a novel missense 631. microcephaly and lessons for neuronal mutation in RBBP8 and a heterozygous Capo‐Chichi J‐M, Bharti SK, Sommers JA, development. Wiley Interdiscip Rev Dev deletion in NRXN1. Gene 538:30–35. Yammine T, Chouery E, Patry L, Rouleau Biol 2:461–478. Ahmad S, TeebiRJG. 1997. Not a new Seckel‐like GA, Samuels ME, Hamdan FF, Michaud JL, Gineau L, Cognet C, Kara N, Lach FP, Dunne J, syndrome but ear‐patella‐short stature syn- Brosh RM Jr, Mégarbane A, Kibar Z. 2013. Veturi U, Picard C, Trouillet C, Eidenschenk drome. Am J Med Genet 70:454. Identification and biochemical characteriza- C, Aoufouchi S, Alcaïs A, Smith O, Arai Y,Pulvers JN, Haffner C, Schilling B, Nusslein tion of a novel mutation in DDX11 causing Geissmann F, Feighery C, Abel L, Smogor- I, Calegari F, Huttner WB. 2011. Neural Warsaw breakage syndrome. Hum Mutat zewska A, Stillman B, Vivier E, Casanova stem and progenitor cells shorten S‐phase on 34:103–107. J‐L, Jouanguy E. 2012. Partial MCM4 commitment to neuron production. Nat Carney JP, Maser RS, Olivares H, Davis EM, Le deficiency in patients with growth retarda- Commun 2:154. Beau M, Yates JR III, Hays L, Morgan WF, tion, adrenal insufficiency, and natural killer Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Petrini JHJ. 1998. The hMre11/hRad50 cell deficiency. J Clin Invest 122:821–832. Chessa L, Smorodinsky NI, Prives C, Reiss protein complex and Nijmegen breakage‐ Gorlin R, Cervenka J, Moller K, Horrobin M, Y, Shiloh Y, Ziv Y. 1998. Enhanced syndrome: Linkage of double‐strand break Witkop CJ. 1975. Malformation syndromes. phosphorylation of p53 by ATM in response repair to the cellular DNA damage response. A selected miscellany. Birth Defects Orig to DNA damage. Science 281:1674–1677. Cell 93:477–486. Artic Ser 11:39–50. 14 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

Griffith E, Walker S, Martin C‐A, Vagnarelli P,Stiff chromosome 15. Am J Hum Genet 65: zaev B, Warren‐Perry M, Snape K, Adlard T, Vernay B, Sanna NA, Saggar A, Hamel B, 1465–1469. JW, Barwell J, Berg J, Brady AF, Brewer C, Earnshaw WC, Jeggo PA, Jackson AP, Kang Y‐H, Lee C‐H, Seo Y‐S. 2010. Dna2 on the Brice G, Chapman C, Cook J, Davidson R, O’Driscoll M. 2008. Mutations in pericen- road to Okazaki fragment processing and Donaldson A, Douglas F, Greenhalgh L, trin cause Seckel syndrome with defective genome stability in eukaryotes. Crit Rev Henderson A, Izatt L, Kumar A, Lalloo F, ATR‐dependent DNA damage signaling. Biochem Mol Biol 45:71–96. Miedzybrodzka Z, Morrison PJ, Paterson J, Nat Genet 40:232–236. Kee Yx, Andrea AD. 2012. Molecular pathogene- Porteous M, Rogers MT, Shanley S, Walker Gruber R, Zhou Z, Sukchev M, Joerss T, Frappart sis and clinical management of fanconi L, Eccles D, Evans DG, Renwick A, Seal S, P‐O, Wang Z‐Q. 2011. MCPH1 regulates anemia. J Clin Invest 122:3799–3806. Lord CJ, Ashworth A, Reis‐Filho JS, the neuroprogenitor division mode by Kerzendorfer C, Colnaghi R, Abramowicz I, Antoniou AC, Rahman N. 2011. Germline coupling the centrosomal cycle with mitotic Carpenter G, O’Driscoll M. 2013. Meier– mutations in RAD51D confer susceptibility entry through the Chk1‐Cdc25 pathway. Gorlin syndrome and Wolf–Hirschhorn to ovarian cancer. Nat Genet 43:879–882. Nat Cell Biol 13:1325–1334. syndrome: Two developmental disorders Loveday C, Turnbull C, Ruark E, Xicola RMM, Guernsey DL, Matsuoka M, Jiang H, Evans S, highlighting the importance of efficient Ramsay E, Hughes D, Warren‐Perry M, Macgillivray C, Nightingale M, Perry S, DNA replication for normal development Snape K, Eccles D, Evans DG, Gore M, Ferguson M, LeBlanc M, Paquette J, Patry L, and neurogenesis. DNA Repair 12:637–644. Renwick A, Seal S, Antoniou AC, Rahman Rideout AL, Thomas A, Orr A, McMaster Kim H, D’Andrea AD. 2012. Regulation of DNA N. 2012. Germline RAD51C mutations CR, Michaud JL, Deal C, Langlois S, cross‐link repair by the fanconi anemia/ confer susceptibility to ovarian cancer. Nat Superneau DW, Parkash S, Ludman M, BRCA pathway. Genes Dev 26:1393–1408. Genet 44:475–476. Skidmore DL, Samuels ME. 2011. Muta- Kiyomitsu T, Murakami H, Yanagida M. 2011. Mahmood S, Ahmad W, Hassan M. 2011. tions in origin recognition complex gene Protein interaction domain mapping of Autosomal recessive primary microcephaly ORC4 cause Meier–Gorlin syndrome. Nat human kinetochore protein Blinkin reveals [MCPH]: clinical manifestations, genetic Genet 43:360–364. a consensus motif for binding of spindle heterogeneity and mutation continuum. Han Y‐G, Alvarez‐Buylla A. 2010. Role of assembly checkpoint proteins Bub1 and Orphanet J Rare Dis 6:39. primary cilia in brain development and BubR1. Mol Cell Biol 31:998–1011. Maréchal A, Zou L. 2013. DNA damage sensing cancer. Curr Opin Neurobiol 20:58–67. Kiyomitsu T,Obuse C, Yanagida M. 2007. Human by the ATM and ATR kinases. Cold Spring Hanks S, Coleman K, Reid S, Plaja A, Firth H, Blinkin/AF15q14 is required for chromo- Harb Perspect Biol 5:223–239. Fitzpatrick D, Kidd A, Mehes K, Nash R, some alignment and the mitotic checkpoint Matsumoto Y, Miyamoto T, Sakamoto H, Robin N, Shannon N, Tolmie J, Swansbury through direct interaction with Bub1 and Izumi H, Nakazawa Y, Ogi T, Tahara H, J, Irrthum A, Douglas J, Rahman N. 2004. BubR1. Dev Cell 13:663–676. Oku S, Hiramoto A, Shiiki T, Fujisawa Y, Constitutional aneuploidy and cancer pre- Koch CA, Agyei R, Galicia S, Metalnikov P, Ohashi H, Sakemi Y,Matsuura S. 2011. Two disposition caused by biallelic mutations in O’Donnell P, Starostine A, Weinfeld M, unrelated patients with MRE11A mutations BUB1B. Nat Genet 36:1159–1161. Durocher D. 2004. Xrcc4 physically links and Nijmegen breakage syndrome‐like Heldin C‐H, Westermark B. 1999. Mechanism of DNA end processing by polynucleotide severe microcephaly. DNA Repair 10:314– action and in vivo role of platelet‐derived kinase to DNA ligation by DNA ligase IV. 321. growth factor. Physiol Rev 79:1283–1316. EMBO J 23:3874–3885. Matsuoka S, Ballif BA, Smogorzewska A, McDo- Hemerly AS, Prasanth SG, Siddiqui K, Stillman B. Kumagai A, Lee J, Yoo HY, Dunphy WG. 2006. nald ER III, Hurov KE, Luo J, Bakalarski 2009. Orc1 controls centriole and centro- TopBP1 activates the ATR‐ATRIP com- CE, Zhao Z, Solimini N, Lerenthal Y,Shiloh some copy number in human cells. Science plex. Cell 124:943–955. Y,Gygi SP,Elledge SJ. 2007. ATM and ATR 323:789–793. Kuo AJ, Song J, Cheung P, Ishibe‐Murakami S, substrate analysis reveals extensive protein Herzog K‐H, Chong MJ, Kapsetaki M, Morgan JI, Yamazoe S, Chen JK, Patel DJ, Gozani O. networks responsive to DNA damage. McKinnon PJ. 1998. Requirement for Atm 2012. The BAH domain of ORC1 links Science 316:1160–1166. in ionizing radiation‐induced cell death in H4K20me2 to DNA replication licensing Matsuura S, Ito E, Tauchi H, Komatsu K, Ikeuchi the developing central nervous system. and Meier–Gorlin syndrome. Nature T, Kajii T. 2000. Chromosomal instability Science 280:1089–1091. 484:115–119. syndrome of total premature chromatid Heyer W‐D, Ehmsen KT, Liu J. 2010. Regulation Lancaster MA, Renner M, Martin C‐A, Wenzel D, separation with mosaic variegated aneuploi- of homologous recombination in eukar- Bicknell LS, Hurles ME, Homfray T, dy is defective in mitotic‐spindle checkpoint. yotes. Annu Rev Genet 44:113–139. Penninger JM, Jackson AP, Knoblich JA. Am J Hum Genet 67:483–486. Hickson ID. 2003. RecQ helicases: Caretakers of 2013. Cerebral organoids model human Meindl A, Hellebrand H, Wiek C, Erven V, the genome. Nat Rev Cancer 3:169–178. brain development and microcephaly. Na- Wappenschmidt B, Niederacher D, Freund Hinchcliffe EHSG. 2001. “It takes two to tango”: ture 501:373–379. M, Lichtner P, Hartmann L, Schaal H, Understanding how centrosome duplication Lavin MF.2008. Ataxia‐telangiectasia: From a rare Ramser J, Honisch E, Kubisch C, Wichmann is regulated throughout the cell cycle. Genes disorder to a paradigm for cell signalling and HE, Kast K, Deiszler H, Engel C, Muller‐ Dev 15:1167–1181. cancer. Nat Rev Mol Cell Biol 9:759–769. Myhsok B, Neveling K, Kiechle M, Mathew Hughes CR, Guasti L, Meimaridou E, Chuang C‐ Lee JE, Gleeson JG. 2011. Cilia in the nervous CG, Schindler D, Schmutzler RK, Hanen- H, Schimenti JC, King PJ, Costigan C, Clark system: Linking cilia function and neuro- berg H. 2010. Germline mutations in breast AJL, Metherell LA. 2012. MCM4 mutation developmental disorders. Curr Opin Neurol and ovarian cancer pedigrees establish causes adrenal failure, short stature, and 24:98–105. RAD51C as a human cancer susceptibility natural killer cell deficiency in humans. J Lee Y, Katyal S, Downing SM, Zhao J, Russell gene. Nat Genet 42:410–414. Clin Invest 122:814–820. HR, McKinnon PJ. 2012a. Neurogenesis Mimitou EP, Symington LS. 2009. DNA end Issa L, Mueller K, Seufert K, Kraemer N, Rose- requires TopBP1 to prevent catastrophic resection: Many nucleases make light work. nkotter H, Ninnemann O, Buob M, Kaindl replicative DNA damage in early progeni- DNA Repair 8:983–995. A, Morris‐Rosendahl D. 2013. Clinical and tors. Nat Neurosci 15:819–826. Mirzaa GM, Vitre B, Carpenter G, Abramowicz I, cellular features in patients with primary Lee Y,Shull ER, Frappart PO, Katyal S, Enriquez‐ Gleeson JG, Paciorkowski AR, Cleveland autosomal recessive microcephaly and a novel Rios V, Zhao J, Russell HR, Brown EJ, DW, Dobyns WB, O Driscoll M. 2014. CDK5RAP2 mutation. Orphanet J Rare Dis McKinnon PJ. 2012b. ATR maintains select Mutations in CENPE define a novel kineto- 8:59. progenitors during nervous system develop- chore‐centromeric mechanism for microce- Jaglin XH, Chelly J. 2009. Tubulin‐related cortical ment. EMBO J 31:1177–1189. phalic primordial dwarfism. Human Genet, dysgeneses: Microtubule dysfunction under- Lobrich M, Jeggo PA. 2007. The impact of a Epub ahead of publication. DOI: 10.1007/ lying neuronal migration defects. Trends negligent G2/M checkpoint on genomic s00439‐014‐1443‐3 Genet 25:555–566. instability and cancer induction. Nat Rev Miyoshi K, Kasahara K, Miyazaki I, Shimizu S, Jamieson CR, Govaerts C, Abramowicz MJ. 1999. Cancer 7:861–869. Taniguchi M, Matsuzaki S, Tohyama M, Primary autosomal recessive microcephaly: Loveday C, Turnbull C, Ramsay E, Hughes D, Asanuma M. 2009. Pericentrin, a centroso- Homozygosity mapping of MCPH4 to Ruark E, Frankum JR, Bowden G, Kalmyr- mal protein related to microcephalic ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 15

primordial dwarfism, is required for olfactory Oettinger MA, Sperling K, Jeggo PA, Spranger S, Toutain A, Trembath RC, Voss cilia assembly in mice. FASEB J 23:3289– Concannon P. 2001. DNA ligase IV muta- E, Wilson L, Hennekam R, de Zegher F, 3297. tions identified in patients exhibiting devel- Dorr H‐G, Reis A. 2008. Mutations in the Miyoshi K, Onishi K, Asanuma M, Miyazaki I, opment delay and immunodeficiency. Mol pericentrin (PCNT) gene cause primordial Diaz‐Corrales F,Ogawa N. 2006. Embryon- Cell 8:1175–1185. dwarfism. Science 319:816–819. ic expression of pericentrin suggests universal O’Driscoll M, Jeggo PA. 2006. The role of Riballo E, Kuhne M, Rief N, Doherty AJ, Smith roles in ciliogenesis. Dev Genes Evol 216: double‐strand break repair—Insights from GCM, Recio M‐J, Reis C, Dahm K, Fricke 537–542. human genetics. Nat Rev Genet 7:45–54. A, Krempler A, Parker AR, Jackson SP, Moldovan G‐L, D’Andrea AD. 2009. How the O’Driscoll M, Jeggo PA. 2008. The role of the Gennery AR, Jeggo PA, Lobrich M. 2004. A fanconi anemia pathway guards the genome. DNA damage response pathways in brain pathway of double strand break rejoining Annu Rev Genet 43:223–249. development and microcephaly: Insight from dependent upon ATM, Artemis and proteins Morris‐Rosendahl DJ, Najm J, Lachmeijer AMA, human disorders. DNA Repair 7:1039– locating to g‐H2AX foci. Mol Cell 16:715– Sztriha L, Martins M, Kuechler A, Haug V, 1050. 724. Zeschnigk C, Martin P, Santos M, Vascon- O’Driscoll M, Ruiz‐Perez VL, Woods CG, Jeggo Romaniello R, Arrigoni F, Cavallini A, Tenderini celos C, Omran H, Kraus U, Van der Knaap PA, Goodship JA. 2003. A splicing mutation E, Baschirotto C, Triulzi F, Bassi M‐T, MS, Schuierer G, Kutsche K, Uyanik G. affecting expression of ataxia‐telangiectasia Borgatti R. 2014. Brain malformations and 2008. Refining the phenotype of a‐1a and Rad3‐related protein (ATR) results in mutations in a‐ and b‐tubulin genes: A Tubulin [TUBA1A] mutation in patients Seckel syndrome. Nat Genet 33:497–501. review of the literature and description of with classical lissencephaly. Clin Genet 74: O’Driscoll M. 2012. Diseases associated with two new cases. Dev Med Child Neurol 425–433. defective responses to DNA damage. Cold 56:354–360. Moshous D, Callebaut I, de Chasseval R, Corneo Spring Harb Perspect Biol 4:411–435. Roos WP, Kaina B. 2006. DNA damage‐induced B, Cavazzana‐Calvo M, Le Deist F, Tezcan I, Ogi T, Walker S, Stiff T, Hobson E, Limsirichaikul cell death by apoptosis. Trends Mol Med Sanal O, Bertrand Y, Philippe N, Fischer A, S, Carpenter G, Prescott K, Suri M, Byrd PJ, 12:440–450. de Villartay JP. 2001. Artemis, a novel DNA Matsuse M, Mitsutake N, Nakazawa Y, Rudner AD, Murray AW. 1996. The spindle double‐strand break repair/V[D]J recombi- Vasudevan P, Barrow M, Stewart GS, Taylor assembly checkpoint. Curr Opin Cell Biol nation protein, is mutated in human severe AMR, O’Driscoll M, Jeggo PA. 2012. 8:773–780. combined immune deficiency. Cell 105: Identification of the first ATRIP–deficient Schneider L, Clement CA, Teilmann SC, Pazour 177–186. patient and novel mutations in ATR define a GJ, Hoffmann EK, Satir P, Christensen ST. Moynahan ME, Jasin M. 2010. Mitotic homolo- clinical spectrum for ATR–ATRIP Seckel 2005. PDGFRaa signaling is regulated gous recombination maintains genomic syndrome. PLoS Genet 8:e1002945. through the primary cilium in fibroblasts. stability and suppresses tumorigenesis. Nat Orii KE, Lee Y, Kondo N, McKinnon PJ. 2006. Curr Biol 15:1861–1866. Rev Mol Cell Biol 11:196–207. Selective utilization of nonhomologous end‐ Shah N, Inoue A, Woo Lee S, Beishline K, Mühlhans J, Brandstätter JH, Gießl A. 2011. The joining and homologous recombination Lahti JM, Noguchi E. 2013. Roles of ChlR1 centrosomal protein pericentrin identified at DNA repair pathways during nervous system DNA helicase in replication recovery from the basal body complex of the connecting development. Proc Natl Acad Sci USA DNA damage. Exp Cell Res 319:2244– cilium in mouse photoreceptors. PLoS ONE 103:10017–10022. 2253. 6:e26496. Pilaz L‐J, Patti D, Marcy G, Ollier E, Pfister S, Shaheen R, Faqeih E, Ansari S, Abdel‐Salam G, Murga M, Bunting S, Montana MF, Soria R, Douglas RJ, Betizeau M, Gautier E, Cortay Al‐Hassnan ZN, Al‐Shidi T, Alomar R, Mulero F, Canamero M, Lee Y, McKinnon V, Doerflinger N, Kennedy H, Dehay C. Sogaty S, Alkuraya FS. 2014. Genomic PJ, Nussenzweig A, Fernandez‐Capetillo O. 2009. Forced G1‐phase reduction alters analysis of primordial dwarfism reveals novel 2009. A mouse model of ATR‐Seckel shows mode of division, neuron number, and disease genes. Genome Res 24:291–299. embryonic replicative stress and accelerated laminar phenotype in the cerebral cortex. Shaheen R, Faqeih E, Shamseldin Hanan E, aging. Nat Genet 41:891–898. Proc Natl Acad Sci USA 106:21924–21929. Noche Ramil R, Sunker A, Alshammari Murray JE, Bicknell LS, Yigit G, Duker AL, van Poirier K, Lebrun N, Broix L, Tian G, Saillour Y, Muneera J, Al‐Sheddi T, Adly N, Al‐Dosari Kogelenberg M, Haghayegh S, Wieczorek Boscheron C, Parrini E, ValenceS, Pierre BS, Mohammed S, Megason Sean G, Al‐Husain D, Kayserili H, Albert MH, Wise CA, Oger M, Lacombe D, Genevieve D, Fontana M, Al‐Mohanna F, Alkuraya Fowzan S. Brandon J, Kleefstra T, Warris A, van der E, Darra F, Cances C, Barth M, Bonneau D, 2012. POC1A truncation mutation causes Flier M, Bamforth JS, Doonanco K, Adès L, Bernadina BD, N’Guyen S, Gitiaux C, a ciliopathy in humans characterized by Ma A, Field M, Johnson D, Shackley F, Firth Parent P, des Portes V, Pedespan JM, Legrez primordial dwarfism. Am J Hum Genet H, Woods CG, Nürnberg P, Gatti RA, V, Castelnau‐Ptakine L, Nitschke P, Hieu T, 91:330–336. Hurles M, Bober MB, Wollnik B, Jackson Masson C, Zelenika D, Andrieux A, Francis Shamseldin HE, Elfaki M, Alkuraya FS. 2012. AP. 2014. Extreme growth failure is a F, Guerrini R, Cowan NJ, Bahi‐Buisson N, Exome sequencing reveals a novel Fanconi common presentation of ligase IV deficiency. Chelly J. 2013. Mutations in TUBG1, group defined by XRCC2 mutation. J Med Hum Mutat 35:76–85. DYNC1H1, KIF5C and KIF2A cause Genet 49:184–186. Musacchio A. 2011. Spindle assembly checkpoint: malformations of cortical development and Shen J, Gilmore EC, Marshall CA, Haddadin M, The third decade. Philos Trans Roy Soc B microcephaly. Nat Genet 45:639–647. Reynolds JJ, Eyaid W, Bodell A, Barry B, Biol Sci 366:3595–3604. Ponting CP. 2006. A novel domain suggests a Gleason D, Allen K, Ganesh VS, Chang BS, Noguchi K, Vassilev A, Ghosh S, Yates JL, ciliary function for ASPM, a brain size Grix A, Hill RS, Topcu M, Caldecott KW, DePamphilis ML. 2006. The BAH domain determining gene. Bioinformatics 22:1031– Barkovich AJ, Walsh CA. 2010. Mutations facilitates the ability of human Orc1 protein 1035. in PNKP cause microcephaly, seizures and to activate replication origins in vivo. EMBO Qvist P, Huertas P, Jimeno S, Nyegaard M, Hassan defects in DNA repair. Nat Genet 42:245– J 25:5372–5382. MJ, Jackson SP, Børglum AD. 2011. CtIP 249. Novorol C, Burkhardt J, WoodKJ, Iqbal A, Roque mutations cause Seckel and Jawad syn- Shiloh Y, Ziv Y. 2013. The ATM protein kinase: C, Coutts N, Almeida AD, He J, Wilkinson dromes. PLoS Genet 7:e1002310. regulating the cellular response to genotoxic CJ, Harris WA. 2013. Microcephaly models Rakic P.1995. A small step for the cell, a giant leap stress, and more. Nat Rev Mol Cell Biol in the developing zebrafish retinal neuro- for mankind: A hypothesis of neocortical 14:197–210. epithelium point to an underlying defect expansion during evolution. Trends Neuro- Sir J‐H, Barr AR, Nicholas AK, Carvalho OP, in metaphase progression. Open Biol 3: sci 18:383–388. Khurshid M, Sossick A, Reichelt S, D’Santos e130065. Rauch A, Thiel CT, Schindler D, Wick U, Crow C, Woods CG, Gergely F. 2011. A primary O’Driscoll M. 2009. Mouse models for ATR YJ, Ekici AB, van Essen AJ, Goecke TO, Al‐ microcephaly protein complex forms a ring deficiency. DNA Repair 8:1333–1337. Gazali L, Chrzanowska KH, Zweier C, around parental centrioles. Nat Genet 43: O’Driscoll M, Cerosaletti KM, Girard P‐M, Dai Y, Brunner HG, Becker K, Curry CJ, Dalla- 1147–1153. Stumm M, Kysela B, Hirsch B, Gennery A, piccola B, Devriendt K, Dorfler A, Kinning Sirbu BM, Cortez D. 2013. DNA damage Palmer SE, Seidel J, Gatti RA, Varon R, E, Megarbane A, Meinecke P, Semple RK, response: Three levels of DNA repair 16 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

regulation. Cold Spring Harb Perspect Biol Tan X, Shi S‐H. 2013. Neocortical neurogenesis Waltes R, Kalb R, Gatei M, Kijas AW, Stumm M, 5:183–198. and neuronal migration. Wiley Interdiscip Sobeck A, Wieland B, Varon R, Lerenthal Y, Sokka M, Parkkinen S, Pospiech H, Syväoja J. Rev Dev Biol 2:443–459. Lavin MF, Schindler D, Dörk T. 2009. 2010. Function of TopBP1 in genome Thornton GK, Woods CG. 2009. Primary Human RAD50 deficiency in a Nijmegen stability. In: Nasheuer H‐P editor. Genome microcephaly: Do all roads lead to Rome? breakage syndrome‐like disorder. Am J Hum stability and human diseases. Houten Trends Genet 25:501–510. Genet 84:605–616. (Utrecht), Netherlands: Springer. pp 119– van der Burg M, Ijspeert H, Verkaik NS, Turul T, Weedon MN, Ellard S, Prindle MJ, Caswell R, 141. Wiegant WW, Morotomi‐Yano K, Mari P‐ Allen HL, Oram R, Godbole K, Yajnik Spalding KL, Bhardwaj RD, Buchholz BA, Druid O, Tezcan I, Chen DJ, Zdzienicka MZ, van CS, Sbraccia P, Novelli G, Turnpenny P, H, Frisén J. 2005. Retrospective birth dating Dongen JJM, van Gent DC. 2009. A DNA‐ McCann E, Goh KJ, Wang Y, Fulford J, of cells in humans. Cell 122:133–143. PKcs mutation in a radiosensitive T–B– McCulloch LJ, Savage DB, O’Rahilly S, Kos Spassky N, Han YG, Aguilar A, Strehl L, SCID patient inhibits Artemis activation and K, Loeb LA, Semple RK, Hattersley AT. Besse L, Laclef C, Romaguera Ros M, nonhomologous end‐joining. J Clin Invest 2013. An in‐frame deletion at the polymer- Garcia‐Verdugo JM, Alvarez‐Buylla A. 2008. 119:91–98. ase active site of POLD1 causes a multisystem Primary cilia are required for cerebellar van der Lelij P, Chrzanowska KH, Godthelp BC, disorder with lipodystrophy. Nat Genet 45: development and Shh‐dependent expansion Rooimans MA, Oostra AB, Stumm M, 947–950. of progenitor pool. Dev Biol 317:246– Zdzienicka MZ, Joenje H, de Winter JP. Weinfeld M, Mani RS, Abdou I, Aceytuno RD, 259. 2010. Warsaw breakage syndrome, a cohe- Glover JNM. 2011. Tidying up loose ends: Stewart GS, Maser RS, Stankovic T, Bressan DA, sinopathy associated with mutations in the The role of polynucleotide kinase/phospha- Kaplan MI, Jaspers NGJ, Raams A, Byrd PJ, XPD helicase family member DDX11/ tase in DNA strand break repair. Trends Petrini JHJ, Taylor AMR. 1999. The DNA ChlR1. Am J Hum Genet 86:262–266. Biochem Sci 36:262–271. double‐strand break repair gene hMRE11 Varon R, Vissinga C, Platzer M, Cerosaletti KM, Woodbine L, Neal JA, Sasi N‐K, Shimada M, is mutated in individuals with an ataxia‐ Chrzanowska KH, Saar K, Beckmann G, Deem K, Coleman H, Dobyns WB, Ogi T, telangiectasia‐like disorder. Cell 99:577– Seemanova E, Cooper PR, Nowak NJ, Meek K, Davies EG, Jeggo PA. 2013. 587. Stumm M, Weemaes CMR, Gatti RA, PRKDC mutations in a SCID patient with Stiff T,Alagoz M, Alcantara A, Outwin E, Brunner Wilson RK, Digweed M, Rosenthal A, profound neurological abnormalities. J Clin HG, Bongers EMHF, O’Driscoll M, Jeggo Sperling K, Concannon P, Reis A. 1998. Invest 123:2969–2980. PA. 2013. Deficiency in origin licensing Nibrin, a novel DNA double‐strand break Woods CG, Parker A. 2013. Investigating micro- proteins impairs cilia formation: Implications repair protein, is mutated in Nijmegen cephaly. Arch Dis Child 98:707–713. for the aetiology of Meier–Gorlin syndrome. breakage syndrome. Cell 93:467–476. Wu Q, Liu J, Fang A, Li R, Bai Y, Kriegstein A, PLoS Genet 9:e1003360. VerloesA, Drunat S, Gressens P,Passemard S. 2013. Wang X. 2014. The dynamics of neuronal Stokes MP,Rush J, MacNeill J, Ren JM, Sprott K, Primary Autosomal recessive microcephalies migration. In: Nguyen L, Hippenmeyer S, Nardone J, Yang V, Beausoleil SA, Gygi SP, and Seckel syndrome spectrum disorders. In: editors. Cellular and molecular control of Livingstone M, Zhang H, Polakiewicz RD, Pagon RA, Adam MP, Bird TD, Dolan CR, neuronal migration. Houten (Utrecht), Comb MJ. 2007. Profiling of UV‐induced Fong CT, Smith RJH, Stephens K, editors. Netherlands: Springer. pp 25–36. ATM/ATR signaling pathways. Proc Natl GeneReviews[R]. Seattle, WA: University of Zimmerman WC, Sillibourne J, Rosa J, Doxsey SJ. Acad Sci USA 104:19855–19860. Washington. PMID20301771. 2004. Mitosis‐specific Anchoring of g Symington LS, Gautier J. 2011. Double‐strand Walczak CE, Cai S, Khodjakov A. 2010. Mech- tubulin complexes by pericentrin controls break end resection and repair pathway anisms of chromosome behaviour during spindle organization and mitotic entry. Mol choice. Annu Rev Genet 45:247–271. mitosis. Nat Rev Mol Cell Biol 11:91–102. Biol Cell 15:3642–3657.