Many roads lead to primary autosomal recessive microcephaly

Progress in Neurobiology 90 (2010) 363–383

Many roads lead to primary autosomal recessive microcephaly

Angela M. Kaindl a,b,c,d,1,*, Sandrine Passemard a,b,e,f,1, Pavan Kumar g, Nadine Kraemer c,d, Lina Issa c,d,Angelika Zwirner c,d, Benedicte Gerard e, Alain Verloes e, Shyamala Mani g,h, Pierre Gressens a,b,f,i

a Inserm, U676, Paris, Franceb Universite Paris 7, Faculte de Medecine Denis Diderot, IFR02 and IFR25, Paris, Francec Department of Pediatric Neurology, Charite – Universitatsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germanyd Institute for Neuroanatomy and Cell Biology, Charite – Universitatsmedizin Berlin, Campus Mitte, Berlin, Germanye APHP, Department of Clinical Genetics, Hopital Robert Debre, Paris, Francef APHP, Department of Pediatric Neurology, Hopital Robert Debre, Paris, Franceg National Brain Research Centre, Manesar, Indiah CNS, IISc, Bangalore, Indiai Institute for Reproductive and Developmental Biology, Imperial College, Hammersmith Campus, London, United Kingdom

A R T I C L E I N F O

Article history:

Received 13 July 2009

Received in revised form 27 October 2009

Accepted 11 November 2009

Keywords:

Microcephaly

MCPH

Centrosome

Spindle

Cell cycle

Mental retardation

A B S T R A C T

Autosomal recessive primary microcephaly (MCPH), historically referred to as Microcephalia vera, is a

genetically and clinically heterogeneous disease. Patients with MCPH typically exhibit congenital

microcephaly as well as mental retardation, but usually no further neurological findings or

malformations. Their microcephaly with grossly preserved macroscopic organization of the brain is a

consequence of a reduced brain volume, which is evident particularly within the cerebral cortex and thus

results to a large part from a reduction of grey matter. Some patients with MCPH further provide evidence

of neuronal heterotopias, polymicrogyria or cortical dysplasia suggesting an associated neuronal

migration defect. Genetic causes of MCPH subtypes 1–7 include mutations in genes encoding

microcephalin, cyclin-dependent kinase 5 regulatory associated protein 2 (CDK5RAP2), abnormal

spindle-like, microcephaly associated protein (ASPM), centromeric protein J (CENPJ), and SCL/TAL1-

interrupting locus (STIL) as well as linkage to the two loci 19q13.1–13.2 and 15q15–q21. Here, we provide

a timely overview of current knowledge on mechanisms leading to microcephaly in humans with MCPH

and abnormalities in cell division/cell survival in corresponding animal models. Understanding the

pathomechanisms leading to MCPH is of high importance not only for our understanding of physiologic

brain development (particularly of cortex formation), but also for that of trends in mammalian evolution

with a massive increase in size of the cerebral cortex in primates, of microcephalies of other etiologies

including environmentally induced microcephalies, and of cancer formation.

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Abbreviations: 53BP1, tumor-suppressor protein p53 binding protein 1; APC, anaphase-promoting complex; ASPM, abnormal spindle-like, microcephaly associated protein;

ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and RAD3-related protein; BACH1, BRCA1-associated carboxyl-terminal helicase; BRCT, carboxyl-terminal

domain of the breast cancer gene 1 BRCA1; BRIT1, BRCT-Repeat Inhibitor of hTert expression; BUBR1, budding uninhibited by benzimidazoles 1 homolog beta; C48, CDK5

activator binding protein C48; CASK, calcium/calmodulin-dependent serine protein kinase gene; CDC24A, cell division cycle 25A; CDK5, cyclin-dependent kinase 5; CDK5R1,

cyclin-dependent kinase 5 regulatory subunit 1; CDK5RAP2, cyclin-dependent kinase 5 regulatory associated protein 2; CENPJ, centromeric protein J; CEP215, centrosome

associated protein 215; CH, calponin homology domain; CHK1/2, checkpoint kinase 1/2; CMD, calmodulin; cnn, centrosomin; CNS, central nervous system; CPAP,

centrosomal P4.1-associated protein; E, embryonal day; EB1, plus-end binding protein EB1; GLI1, glioma-associated oncogene homolog; gTuRC, gamma-tubulin ring

complex; Hnf3b, hepatocyte nuclear factor 3-beta, forehead box A2; LAP, LAG-3-associated protein; Lefty2, left–right determination factor 2; LIP1, LYST-interacting protein 1;

MAD2, mitotic arrest-deficient 2; MCPH, autosomal recessive primary microcephaly; MCPH1, microcephalin; MDC1, mediator of DNA damage checkpoint protein 1; MOT,

microtubule-organizing center; NBS1, Nijmegen breakage syndrome protein 1; NE, neuroepithelium; OFC, occipito-frontal head circumference; PCC, premature chromosome

condensation; PCM, pericentriolar matrix; PCNT, pericentrin; Pitx2, paired-like homeodomain transcription factor 2; PLK1, polo-like kinase 1; RELN, reelin; Sas-4, spindle

assembly abnormal 4; SD, standard deviation; Shh, sonic hedgehog; shRNA, short hairpin RNA; STIL, SCL/TAL1-interrupting locus; SUFU, Suppressor of Fused; Tbr1, T-box

brain 1; TNFalpha, tumor necrosis factor-alpha; zyg-1, ZYGote defective, embryonic lethal.

* Corresponding author at: Department of Pediatric Neurology, Charite – University Medicine Berlin, Campus Virchow Klinikum, 13353 Berlin, Germany.

Tel.: +49 030 450 566 112; fax: +49 030 450 566 920.

E-mail address: [email protected] (A.M. Kaindl).1 These authors contributed equally to this work.

0301-0082/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pneurobio.2009.11.002

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A.M. Kaindl et al. / Progress in Neurobiology 90 (2010) 363–383364

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

2. Phenotype of patients with MCPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

3. Genotype of patients with MCPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

4. Pathomechanisms underlying MCPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

5. Microcephalin (syn. BRIT1; MCPH1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

6. Cyclin-dependent kinase 5, regulatory associated protein 2 (syn. CDK5RAP2, C48, CEP215, KIAA1633, MCPH3) . . . . . . . . . . . . . . . . . . . . . 373

7. Abnormal spindle-like, microcephaly-associated (ASPM, MCPH5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

8. Centromeric protein J (syn. CENPJ, CPAP, LAP, LIP1, MCPH6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378

9. SCL/TAL1-interrupting locus (syn. STIL, SIL, MCPH7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378

10. MCPH and cerebellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

11. The role of MCPH proteins in the evolution of the cerebral cortex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

12. Other proteins that show an overlap with MCPH pathomechanisms and phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

13. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

1. Introduction

Microcephaly is defined as a small cranium with a significantlyreduced occipito-frontal head circumference (OFC) of more thantwo standard deviations (SD) below the mean for age, sex, andethnicity (severe microcephaly OFC < �3 SD). Microcephaly can beacquired (caused by environmental factors) or hereditary in originand can become apparent congenitally (primary microcephaly) orpostnatally (secondary microcephaly). The incidence of micro-cephaly at birth, as evaluated in birth defect registers world-wide,varies from 1.3 to 150 per 100,000 live-births, depending on thepopulation and the applied SD threshold to define microcephaly(source: International Clearinghouse for Birth Defects Surveillanceand Research, 2006 report; http://www.icbdsr.org/). Primary, non-syndromal microcephaly has an incidence of 1:30,000 to 1:250,000live-births (Van Den Bosch, 1959).

Microcephalia vera (‘true microcephaly’) is a loosely defined,historical term referring to children with isolated, non-syndromalcongenital microcephaly. This term was coined without consider-ation of etiology or neuropathology, and it is still applied, however,in a narrower sense, to designate patients with non-syndromalautosomal recessive microcephaly without lissencephaly orpachygyria. Syndromic microcephalies as well as secondary, oftenenvironmentally induced microcephalies which can be caused byunderlying processes such as high rates of pathologic apoptosis,proliferation and patterning abnormalities, migration defects,disturbance of extracellular matrix integrity and defects ofsynaptogenesis will not be discussed in this review (please referto Francis et al., 2006; Katyal and McKinnon, 2007; Abuelo, 2007;Woods, 2004).

2. Phenotype of patients with MCPH

Autosomal recessive primary microcephaly (MCPH, for Micro-Cephaly Primary Hereditary) is a rare, genetically heterogeneousdisease reported in about 100 families world-wide. Historically,primary microcephaly was defined by an OFC < �3 at birth, areduced brain volume, mental retardation (IQ between 30 and 70–80) and no further neurological findings except for mild seizures(Woods et al., 2005; Passemard, 2009) (Fig. 1). However, it isbecoming clear that the true phenotype spectrum of patients withMCPH gene mutations is wider than indicated by previouspublications which for the most part provide no detailedphenotype information. In individual patients, the OFC is stillin the normal range (around �2 SD) at birth followed by adevelopment of a microcephaly within the first year of life(Passemard, 2009). As recently noted, MCPH may already be

evident by the 24th week of gestation through ultrasound and/orMRI analysis (Tunca et al., 2006). Also, we were able todemonstrate recently that neurological features can indeed occurin patients with MCPH due to ASPM gene mutations (Passemard,2009). These include speech delay, hyperactivity and attentiondeficit, aggressiveness, focal or generalized seizures, delay ofdevelopmental milestones and pyramidal signs (Passemard, 2009).Hyperactivity and attention deficit appeared to be major childhoodproblems in all patients of our cohort and possibly causedimpairments in performance. Moreover, abnormal height andweight was detected in some patients (Passemard, 2009; Trimbornet al., 2004).

Imaging studies reveal typically brains of ‘normal architecture’but of reduced size (Woods et al., 2005). The latter is particularlyevident in the cerebral cortex, which shows a simplified cerebralcortex structure, and there is also a slightly reduced white mattervolume (Fig. 2). Individual patients with MCPH provide evidenceof periventricular neuronal heterotopias (Woods et al., 2005;Trimborn et al., 2004) suggesting neuronal migration defects.Moreover, we have detected infra-tentorial anomalies (brainstemor cerebellar hypoplasia), dysmorphic and/or enlarged lateralventricles and corpus callosum agenesia as well as focal micro-polygyria and/or dysplasia (Passemard, 2009). Future studies willneed to address in what way white matter disease also contributesto brain size reduction in MCPH patients. Descriptions of thehistological findings in MCPH patients, indicating a significantlyreduced brain volume with an almost preserved convolutionpattern, are rare and were performed at a time when a geneticdiagnosis was not possible (Bamatter and Rabinowicz, 1969;Robain and Lyon, 1972). An overview of MCPH features, genemutations and the reported phenotype by MCPH gene mutationare given in Tables 1–3, respectively.

3. Genotype of patients with MCPH

Genetic causes of MCPH subtypes 1–7 include mutations ingenes encoding microcephalin (MCPH1; Jackson et al., 2002, 1998;OMIM #251200), cyclin-dependent kinase 5 regulatory associatedprotein 2 CDK5RAP2 (MCPH3; Bond et al., 2005; Moynihan et al.,2000; OMIM #604804), abnormal spindle-like, microcephalyassociated ASPM (MCPH5; Pattison et al., 2000; Shen et al.,2005; OMIM #608716), centromeric protein J CENPJ (MCPH6;Bond et al., 2005; Leal et al., 2003; OMIM #608393), SCL/TAL1-interrupting locus STIL (MCPH7; Kumar et al., 2009; OMIM#612703) as well as linkage to the two loci 19q13.1–13.2 (MCPH2;Roberts, 1999); OMIM%604317) and 15q15–q21 (MCPH4; Jamie-son et al., 1999); OMIM%604321) (Kaindl, 2008); see Table 2 and

http://www.icbdsr.org/

omim:251200

omim:604804

omim:608716

omim:608393

omim:612703

omim:604317

omim:604321

Fig. 1. Typical clinical features of a patient with MCPH. Clinical evaluation of patients with MCPH typically reveals a microcephaly. (a and b) Note the typical flattening of the

forehead of this 4-y-old girl carrying a compound mutation of the ASPM gene mutation (c.2389 C > T [p.Arg797X] and c.4074 G > A [p.Trp1358X]). (c) Her OFC was below 5 SD

at birth and further decreased to about �9 SD at the age of 7 y.

A.M. Kaindl et al. / Progress in Neurobiology 90 (2010) 363–383 365

Fig. 3A–E. Further genetic heterogeneity likely exists as, in ourexperience, about 20–30% of families with MCPH do not showlinkage to any of the currently known loci. Most reportedmutations induce the production of non-functional, truncatedproteins. MCPH genes are highly conserved among species and aresuspected to have played a critical role in brain evolution (forreview, see Woods et al., 2005; Gilbert et al., 2005; Ponting andJackson, 2005).

The phenotype of humans with MCPH and of animal modelsindicates that pathomechanisms underlying MCPH include adecrease or abnormal cell proliferation/differentiation, increasedrates of cell death and possibly neuronal migration defects.

4. Pathomechanisms underlying MCPH

Brain size at birth is largely determined by the relative rates ofproliferation and cell death occurring during neurogenesis. Neuralprogenitors are generated by symmetric and asymmetric celldivisions (Fig. 4), and cell death can be induced by defects of cellcycle regulation/abnormal DNA damage response (Fig. 5). Thus, animpairment of neural progenitor divisions and/or DNA repair couldbe responsible for a marked reduction of the cortical area. MCPH-associated genes encode proteins implicated in cell cycle regula-tion, cell cycle checkpoint control and DNA repair, chromosomecondensation, centrosome function, spindle formation and dy-namics, kinetochore attachment to spindles, cellular abscissionand apoptosis. They localize predominantly to the centrosome andthe pericentriolar matrix in tumor cells (Fig. 6). Their localizationin neural cells has not been reported in detail. In the following, weprovide a critical overview of current knowledge on MCPH gene

product function, mechanisms leading to microcephaly in humanswith MCPH and abnormalities in cell division/cell survival incorresponding animal models.

5. Microcephalin (syn. BRIT1; MCPH1)

The first mutations detected in patients with MCPH were withinthe MCPH1 gene which encodes the protein microcephalin, alsoreferred to as BRIT1 for BRCT-Repeat Inhibitor of hTert expression(Jackson et al., 2002, 1998; Lin and Elledge, 2003). The full length,835 amino acids human microcephalin protein has one N-terminaland two C-terminal BRCT domains (BRCT = carboxyl-terminaldomain of the breast cancer gene 1 BRCA1, Fig. 3A). Orthologgenes with similar domain structures are also found in modelorganisms such as the mouse, Drosophila melanogaster andCaenorhabditis elegans (Ponting and Jackson, 2005). MicrocephalinmRNA is expressed in human fetal tissues, especially in brain, liverand kidney, as well as in a number of adult tissues, as detected byRT-PCR (Jackson et al., 2002). In the fetal murine brain,microcephalin was found to be expressed during neurogenesiswith high levels in the forebrain and within proximity to the lateralventricles using in situ hybridization; the latter region holdsprogenitor cells that produce neurons which migrate and form thecerebral cortex (Jackson et al., 2002). Loss of function causesMCPH1, microcephaly with simplified gyral pattern and prematurechromosome condensation with microcephaly and mental retar-dation (PCC) in humans (MIM*607,117).

The human phenotype due to MCPH1 mutations may be causedby defective cell cycle checkpoint control and DNA repair. Cellcycle checkpoints are regulatory pathways that govern the order

Fig. 2. Radiological features of patients with MCPH. Cranial MRI of the patient depicted in Fig. 1 with an ASPM gene mutation (A) compared to an age matched control (B). Axial

(1) and coronal (2) T2-weighted images show the typical reduction of brain volume, especially of cerebral cortex in a patient with MCPH (A) in comparison to an age-matched

control (B). Note the typical appearance of a simplified gyral pattern with scarce gyri in the patient. Here, the number of gyri is reduced and the sulci are shallow compared to

the control person. Sagittal (3) T1-weighted images illustrate the typical MRI features of patients with ASPM mutations in comparison to controls. Note the poorly developed

frontal lobes in the patient and the agenesis of the rostrum of the corpus callosum (white arrow).


and timing of cell cycle transitions to ensure completion of onecellular event prior to commencement of another one. Thesecheckpoint control mechanisms are linked to those of DNA damagerepair as, in response to DNA damage, the cell cycle needs to bedelayed until the damage is repaired to restore the integrity of theorganism or, if repair is not possible, arrested with a subsequentinduction of cell death. The BRCT domains occurring in MCPH1 areevolutionarily conserved phosphor-peptide binding amino acidtandem repeat domains involved in cell cycle control (Bork et al.,1997; Huyton et al., 2000; Yu et al., 2003). The BRCT domain wasfirst identified within the tumor-suppressor gene product BRCA1(Yu et al., 2003); there it interacts with phosphorylated BRCA1-associated carboxyl-terminal helicase (BACH1) in a cell cycleregulated manner, and this interaction is required for DNAdamage-induced checkpoint control during the transition fromG2 phase to M phase of the cell cycle (Yu et al., 2003). Genomicmutations of BRCA1 as well as reduced expression can be found infamilial breast cancer and other tumor specimen (Yu et al., 2003;Rai et al., 2006; Hagemann et al., 2008).

Microcephalin is induced by DNA damage and forms nuclearfoci minutes after irradiation-induced DNA damage in tumor cells(Rai et al., 2006). The protein is recruited to double-strand breaksvia interaction of its C-terminal BRCT domains with phosphory-lated H2AX, a histone variant that marks sites of damaged DNA(Wood et al., 2007). This then allows for recruitment of othermediator proteins that help promote the damage signal to CHK1and CHK2, which can phosphorylate various substrates tomodulate the cell cycle, DNA repair and apoptosis (Wood et al.,2007). Microcephalin specifically co-localizes to and activates DNAdamage response proteins, such as mediator of DNA damagecheckpoint protein 1 (MDC1), tumor-suppressor protein p53binding protein 1 (53BP1), Nijmegen breakage syndrome protein1 (NBS1) and phosphorylated ATM (ataxia telangiectasia, mutated)(Rai et al., 2006). Mutated MCPH1-transcript detected in thebreast cancer specimen was unable to complement the defectiveactivation of DNA damage response proteins, in contrast to fulllength MCPH1 (Rai et al., 2006). MCPH1 interacts with condensin IIin the homologous repair of DNA damage (Wood, 2008). Moreover,

Table 1Overview of MCPH features.

Main features � Microcephaly (�2 SD) at birth, further

‘relative’ reduction of OFC in the first years of life

� Reduction of cerebral cortex volume

� Simplification of gyral pattern

� Mild to severe mental retardation

(normal IQ possible)

Inconsistent features � Neurological/neuropsychological

- Delay of motor milestones

- Pyramidal signs

- Speech delay

- Hyperactivity and attention deficit

- Aggressiveness

- Sleep disorder

- Seizures (tonic-clonic, focal, generalized)

� Further intracranial malformations

- Agenesis of the corpus callosum

- Focal dysplasia

- Focal microgyria

- Perisylvanian polymicrogyria

- Dysmorphic or enlarged lateral ventricles

- Large pituitary gland

- Reduction of white matter

- Infra-tentorial anomalies (brainstem/

cerebellar hypoplasia)

� Endocrinology

- Short stature

- Early puberty

� Extracranial malformations

- Syndactyly

- Renal agenesia

- Multicystic kidney

� Other

- Premature chromosome condensation

(e.g. in MCPH1)

- So far no increased cancer risk noted


MCPH1 gene mutations result in premature onset of condensin II-mediated chromosome condensation (Trimborn et al., 2006).Further evidence that MCPH1 may be involved in checkpointcontrol comes from experiments using RNA interference. Suchan approach lead to a severe impairment of the intra-S phase andG2/M DNA damage checkpoint in U2OS cells (human osteosarcomacell line) with a loss of checkpoint control allowing cells to proceedinto mitosis despite exposure to considerable doses of ionizingirradiation (IR), an increased sensibility to DNA damage via IR and adecrease of BRCA1 and the checkpoint kinase 1 (CHK1) (Xu et al.,2004; Lin et al., 2005). The latter are key regulators in the control ofintra-S and G2/M checkpoints. MCPH1 also acts downstream fromCHK1 in regulating the stability of cell division cycle 25A (Cdc25A),a critical regulator of cell cycle progression, and consequentlypreventing premature entry into mitosis (Alderton et al., 2006).Microcephalin may thus affect DNA damage checkpoints by bothdirectly transmitting DNA damage signals and controlling theexpression levels of other checkpoint regulators. It has recentlybeen shown that Microcephalin promotes the expression of CHK1

Table 2Overview of MCPH genes.

MCPH Protein Gene

MCPH1 Microcephalin MCPH

MCPH2 –

MCPH3 Cyclin-dependent kinase 5 regulatory associated protein 2 CDK5

MCPH4 –

MCPH5 Abnormal spindle-like, microcephaly associated ASPM

MCPH6 Centromeric protein J CENP

MCPH7 SCL/TAL1 interrupting locus STIL

and BRCA1 through the interaction with the transcription factorE2F1 and enhancement of their transcriptional activities on thepromoters of both genes as well as other E2F targeted genesinvolved in DNA repair and apoptosis such as RAD51, DDB2,TOPBP1, p73 and caspases (Yang et al., 2008). In humanlymphoblastoid cell lines with MCPH1 truncating mutations, lossof MCPH1 function induces defective G2/M checkpoint arrest,nuclear fragmentation after DNA damage and supernumerarymitotic centrosomes (Alderton et al., 2006).

Several findings underline the role of MCPH1 as a regulator ofchromosome condensation. Mutations in the MCPH1 gene have notonly been reported in patients with MCPH but also in those withpremature chromosome condensation (PCC) syndrome, character-ized by microcephaly, short stature and misregulated chromosomecondensation (Trimborn et al., 2004). Intriguingly, the cellularphenotype of patients with PCC or MCPH1 includes high numbersof prophase-like cells due to premature chromosome condensationin early G2-phase and delayed decondensation in post-mitosis.This cellular phenotype can be reproduced through MCPH1-siRNAmediated reduction of the mRNA (Trimborn et al., 2004, 2006).MCPH1 inhibits condensin II, a protein important for chromosomearchitecture, potentially via CDK-cyclin A (Trimborn et al., 2006).MCPH1 appears to also influence centrosome function as itlocalizes to the centrosome throughout the cell cycle in U2OScells (Zhong et al., 2006) and also in other model organisms (Brunket al., 2007; Rickmyre et al., 2007; Jeffers et al., 2008). The N-terminal BRCT1 has been reported to be required for centrosomallocalization in irradiated cells (Jeffers et al., 2008). Knock-down ofMicrocephalin in U2OS cells via siRNA induced centrosomeanomalies (possibly a secondary effect), spindle misalignment aswell as compacted and lagging chromosomes with defectivespindle checkpoint activation and delayed cytokinesis by nega-tively regulating Aurora and polo-like kinase 1 (PLK1) (Rai et al.,2008). Spindle positioning is critical to ensure the unequalsegregation of polarity factors and generate daughter cells withdifferent sizes or fates during asymmetric cell division. Treatmentof MCPH1 patient lymphoblastoid cell lines with nocodazoleresults in supernumerary centrosomes (Alderton et al., 2006).Mcph1 appears to be essential for the coordination of mitosis in theDrosophila embryo as, in mcph1 mutant flies, mitotic entry isslowed from the very first mitosis with prolonged prophase andmetaphase stages frequent premature separation as well asdetachment of centrosomes (Brunk et al., 2007; Table 4). As aconsequence, centrosome and nuclear cycles become uncoordi-nated, resulting in premature chromosome condensation, mitoticentry with unreplicated DNA, genomic instability and arrestedembryonic development (Brunk et al., 2007; Rickmyre et al.,2007). Brains of adult male mcph1�/� flies are reportedly ofnormal size but have defects in mushroom body structure,suggesting an evolutionarily conserved role for MCPH1 inbrain development (Rickmyre et al., 2007). Microcephaly occurringin MCPH1-deficiency may thus also be due to a defectivecentrosomal function of MCPH1 influencing the number ofproliferative, symmetric cell divisions of neuronal stem cellsduring neurogenesis.

Chromosome References

1 8p23 Jackson et al. (2002, 1998)

19q13.1–q13.2 Roberts (1999)

RAP2 9q33.3 Bond et al. (2005), Moynihan et al. (2000)

15q15–q21 Jamieson et al. (1999)

1q31 Pattison et al. (2000), Shen et al. (2005)

J 13q12.2 Bond et al. (2005), Leal et al. (2003)

1p32 Kumar et al. (2009)

Table 3MCPH phenotype and genotype.

Gene mutation Origin Patient/family OFC at

birth

OFC at

follow up

Mental

retardation

Neurological

phenotype

Neuroradiological

phenotype

Further

phenotype

References

MCPH1

74 C>G Pakistan 7 patients,

2 families

? �5 to �10 SD

(6–28-y-old)

Mild to

moderate

- Inability to read or

write, all could speak

- CT normal in 1 child - Short stature

(�1 to �4 SD

(Trimborn

et al., 2004))*

Trimborn et al. (2004),

Jackson et al.

(2002, 1998)

- Basic self-care skills - Marked reduction of

cerebral cortex size,

cerebellar and brain

stem volume in 1 adult

- Premature

chromosome

condensation

- No focal neurological

deficits or epilepsy

- Microcephaly with

simplified gyri

(Trimborn et al., 2004)

Linked to

MCPH1 locus

Pakistan 7 patients,

2 families

>�4 SD �7 to �10 SD Mild to

moderate

Roberts et al. (2002)

ins 427A Lebanon 2 patients �3.5 to �4 SD �10 to �12 SD Profound - Spasticity - Periventricular neuronal

heterotopias

- Short stature

(�5 SD)

Trimborn et al. (2004),

Neitzel et al. (2002)

- Motor and

speech delay

- Marked gyral

simplification

(pachygyria appearance)

- Premature

chromosome

condensation

- Frontal lobe hypoplasia - Unilateral renal

agenesia

- Agenesis of the

CC genu

80 C>G Caucasian 1 patient �2.4 SD �3 SD Verbal IQ 115,

overall

IQ 84 (6 y)

- Normal

psychomotor skills

? - Short stature Trimborn et al. (2005)

- Premature

chromosome

condensation

- Syndactyly II–III

CDK5RAP2

243T-A North Pakistan 1 family ? Bond et al. (2005)

IVS26–15A North 1 family ? Bond et al. (2005)

Pakistan

243T-A (=246 T>A) North Pakistan 1 family >�4 to �7 SD

(18–30 y)

- Mild to

moderate

(IQ 51–65)

- Slopped forehead,

otherwise no facial

features

Hassan et al. (2007)

- No other neurological

problems

ASPM

74delG Caucasian 1 family ^ Nicholas et al. (2009)

77delG and 6232T>C France 1 patient �2 SD �5 SD IQ 70 - Hyperactivity disorder - Normal Passemard (2009)

- Aggressiveness

- Automutilation (3–6 y)

297 and

1460_3391del21844

Caucasian 1 family ^ Nicholas et al. (2009)

349C>T Pakistan 1 family & - Normal Bond et al. (2003)

s 349C>T India 2 families �12 to �13 SD SQ 31.5–44.5 § - Mild motor delay Kumar et al. (2004)

- Aggressiveness

- Hyperactivity in

1 patient

440delA Caucasian 1 family ^ Nicholas et al. (2009)

577C>T Caucasian 1 family ^ Nicholas et al. (2009)

719delCT Pakistan 1 family ? - Striking reduction

of cerebral cortex

Bond et al. (2002)

- Simplification of

gyral pattern

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36

3–

38

33

68

1002delA Pakistan 1 family Muhammad et al. (2009)

1152delAG Caucasian 1 family ^ Nicholas et al. (2009)

1258delTCTCAAG Pakistan 2 families ? & - Normal Bond et al. (2002, 2003)

1260delTCAAGTC Pakistan 1 family # - Moderate - Normal Gul et al. (2006)

1630delATCTT and

full length ASPM

gene del

France 1 patient �3.5 SD �7 SD IQ 30 - Mild motor and

speech delay

- Simplification of

frontal and occipital

gyral pattern

- Early puberty Passemard (2009)

- Hyperactivity

disorder (3–12 y)

- Enlarged occipital

horns of the LV

1366G>T Turkey 1 family ^ Nicholas et al. (2009)

1406delATCCTAAAA Caucasian 1 family ^ Nicholas et al. (2009)

1590delA Caucasian 1 family ^ Nicholas et al. (2009)

1727delAG Yemen 1 family �10 to �11 SD - Severe - Normal Bond et al. (2003)

1959delCAAA Saudi Arabia 1 family & - Normal Bond et al. (2003),

Nicholas et al. (2009)

Caucasian 1 family


2389C>T and

4074G>A

Morocco 1 patient �5 SD �9 SD IQ 42 - Motor and speech

delay

- Agenesis of CC rostrum Passemard (2009)

- Hyperactivity

disorder

- Enlarged LV

- Coarse gyri

- Simplified frontal and

occipital gyral pattern

- Large pituitary

2389C>T and

6686delTTAAA

Lebanon/

Algeria

1 patient �6 SD �7 SD IQ 42 - Normal motor

development

- Agenesis of CC rostrum Passemard (2009)

- Hyperactivity disorder - Enlarged LV occipital horns

- Pyramidal signs - Simplified gyral pattern

- Sleep disturbance - Posterior focal parietal

dysplasia

2761-25A>G Caucasian 1 family ^ Nicholas et al. (2009)

IVS9 and 5G>T

(2936 and 5G>T)

Pakistan 1 family & - Normal Bond et al. (2003)

2938C>T Pakistan 1 family Muhammad et al. (2009)

2967G>A Caucasian 1 family ^ Nicholas et al. (2009)

3055C>T Caucasian 2 families ^ Nicholas et al. (2009)

3055C>T and

7894C>T

Pakistan� 1 family Muhammad et al. (2009)

3082GA Pakistan 1 family & - Normal Bond et al. (2003)

3188T>G Pakistan 1 family ^ Nicholas et al. (2009)

3477delCGCTA Pakistan 1 family Muhammad et al. (2009)

3527C>G Jordani 1 family & - Normal Bond et al. (2003)

3663delG Pakistan 2 families �11 SD - Normal Bond et al. (2003)

3710C>G Caucasian 1 family ^ Nicholas et al. (2009)

3741 and 1G>A Caucasian 1 family ^ Nicholas et al. (2009)

3796G>T Africa 1 family ^ Nicholas et al. (2009)

3811C>T Germany 2 families & - Normal Bond et al. (2003)

Asia 1 family Nicholas et al. (2009)

Reunion

island

1 patient �5 SD �8 SD - Mild motor delay.

Hyperactivity disorder

- Agenesis of CC splenium Passemard (2009)

- Sleep disturbance

3945delAG and

9191delGAAA

Germany 1 patient �4 SD �7 SD IQ 104 - Normal motor

development

- Simplified frontal

gyral pattern

- Short stature

(�2 SD at birth,

�3 SD at 4 y)

Passemard (2009)

- Enlarged LV occipital

horns

- Dysmorphic frontal

ventricles

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3–

38

33

69

Table 3 (Continued )


birth

OFC at

follow up

Mental

retardation

Neurological

phenotype

Neuroradiological

phenotype

Further

phenotype

References

3978G>A India 1 family �11 SD SQ 48§ - Mild motor and

language delay

Kumar et al. (2004),

Gul et al. (2006)

- Hyperactivity

Pakistan * # - Moderate

3978G>A and

9319C>T

Pakistan 1 family - Seizures in infancy Muhammad et al. (2009)

4581delA Pakistan 1 family & - Normal Bond et al. (2003)


4855del TA Pakistan 1 family ^ Nicholas et al. (2009)

5136C>A Pakistan 1 family ^ Nicholas et al. (2009)

5149delA Pakistan 1 family ^ Nicholas et al. (2009)

6189T>G Saudi Arabia 3 patients

1 family

�4 to �5 SD - Moderate

(IQ 55) to

severe (IQ 35)

- Normal motor

development

- Mild simplification of

cerebral cortical

gyral pattern

Shen et al. (2005)

- Tonic/clonic seizures

(onset at 32–33 mo)

in 2 patients

- Slight reduction of

white matter

- Focal seizures

(left sided epileptic

discharges with right

hemiparesis) in

1 patient

6335delAT Pakistan 1 family ^ Nicholas et al. (2009)

6651del AACA France 1 patient �2.5 SD �3.5 SD IQ 43 - Hyperactivity disorder,

oppositional defiant

disorder (3–6 y)

- Simplification of


gyral pattern

Passemard (2009)

- Enlarged LV

occipital horns

6732delA Pakistan 1 family Muhammad et al. (2009)

7489delTATAT Caucasian 1 family ^ Nicholas et al. (2009)

7761T Pakistan 1 family ? - Normal Bond et al. (2002),


Pakistan 1 family

7782delGA Pakistan 2 families ^ Nicholas et al. (2009)

Caucasian 1 family,

2 patients

�2 SD

Algeria �5 SD �4 SD IQ 70 - Normal - Simplification of the


gyral pattern

Passemard (2009)

�7 SD IQ 53 - Normal

7859delAG Saudi Arabia 1 family ^ - Tonic/clonic

seizures after 1 y




8130delAA Pakistan 1 family ^ Nicholas et al. (2009)

8190delAG +

8273T>A

France 1 patient �2 SD �4.5 SD - Hyperactivity disorder

(3–6 y), very calm >6 y

- Normal - Unilateral

multicystic kidney

Passemard (2009)

8378delT Pakistan 2 families ^ Nicholas et al. (2009)

8508delGA Pakistan 1 family & - Normal Nicholas et al. (2009),

Bond et al. (2003)

Pakistan 1 family


8844delC Caucasian 1 family ^ Nicholas et al. (2009)

9118insCATT Pakistan 1 family # - Moderate - Normal Gul et al. (2006)

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36

3–

38

33

70

9159delA Pakistan 1 family & - Normal Kumar et al. (2002), Zhong et al. (2005),

Fish et al. (2006), van der Voet et al. (2009),

Casal et al. (1990), Fujimori et al. (2008),

Hung et al. (2004), Chen et al. (2006),

Hung et al. (2000), Koyanagi et al. (2005),

Hermeking (2003), Basto et al. (2006),

Stevens et al. (2007), Kirkham et al. (2003),

Leidel and Gonczy (2003),

Dammermann et al. (2008),

Pelletier et al. (2006), Delattre et al. (2006),

Karkera et al. (2002), Aplan et al. (1990),

Aplan et al. (1992), Brown et al. (1990),

Erez et al. (2004), Ramaswamy et al. (2003),

Campaner et al. (2005), Erez et al. (2008),

Izraeli et al. (1997), Erez et al. (2007),

Kasai et al. (2008), Pfaff et al. (2007),

Collazo-Garcia et al. (1995),

Izraeli et al. (1999), Izraeli et al. (2001),

Baala et al. (2007), Najm et al. (2008),

Evans et al. (2004a),

Mekel-Bobrov et al. (2005),

Evans et al. (2006), Evans et al. (2005),

Evans et al. (2004b), Ali and Meier (2008),

Wang (2008), Wang and Su (2004),

Balter (2005), Balter (2006a,b),

Bond and Woods (2006), Cotoi et al. (1980),

Currat (2006), Frost (2008),

Kouprina et al. (2004), Ponting (2006),

Richards (2006), Stern and Woods (2006),

Zhang (2003), Griffith et al. (2008),

Rauch et al. (2008), Tibelius (2009),

Roberts et al. (2002), Neitzel et al. (2002),

Trimborn et al. (2005), Hassan et al. (2007),

Nicholas et al. (2009),

Bond et al. (2002, 2003)

9178C>T India 2 families �8 to �9 SD SQ 50–62§ - Mild motor delay,

aggressiveness,

hyperactivity

in 1 patient

Kumar et al. (2004)

Caucasian 1 family Nicholas et al. (2009)

9190C>T Pakistan 1 family & - Normal Nicholas et al. (2009),

Bond et al. (2003)

Germany 1 family

9238A>T transition Pakistan 1 family # Mild - Normal Gul et al. (2006)


9319C>T +

9507delG

France 1 family with

2 patients

�4 SD �6 SD IQ 55 - Normal skills

development

- Simplified frontal

and occipital gyral

pattern

Passemard (2009)

�4 SD �5 SD IQ 46 - Hyperactivity disorder

(<6 y), apathy (>8 y)

- Enlarged LV

- Focal epilepsy

(>14 y; polymicrogyria

patient)

- Extensive unilateral

perisylvian

polymicrogyria

9492T>G Pakistan 1 family Muhammad et al. (2009)

9539A>C

substitution

Pakistan 1 family # Mild to

moderate

- Normal Gul et al. (2006)

9557C>G Pakistan 2 families* # - Mild to

moderate

- Normal Bond et al. (2003),

Gul et al. (2006)

Pakistan

9595A>T Pakistan 1 family Muhammad et al. (2009)

9677insG Pakistan 1 family Muhammad et al. (2009)

9681delA Pakistan 1 family ^ Nicholas et al. (2009)

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3–

38

33

71

Table 3 (Continued )


birth

OFC at

follow up

Mental

retardation

Neurological

phenotype

Neuroradiological

phenotype

Further

phenotype

References

9686delTTAAA Lebanon 1 family,

4 patients

�7 to �8 SD - Profound - Motor and speech

delay

Passemard (2009)

- Generalized

tonic/clonic seizures

(onset 18 mo and 8 y)

9697C>T Pakistan� Muhammad et al. (2009)

9730C>T Pakistan ^ Nicholas et al. (2009)

9754delA Yemen 1 family �10 to �11 SD - Severe - Normal Bond et al. (2003)

9789T>A Pakistan 1 family ^ Nicholas et al. (2009)

IVS25 + 1G>T

(9984 + 1G>T)

Pakistan 1 family �7 SD - Normal Bond et al. (2003)

IVS25 + 1G>T

(9984 + 1G>T)/

3663delG

Pakistan 1 patient �9 SD - Profound Bond et al. (2003)

10,059C>A Pakistan 1 family ^ Nicholas et al. (2009)

Translocation

(1;4)(q31;p15.3)

? 1 patient Pichon et al. (2004)

large deletion

21.844b (loss of

exons2 to 13)

1 patient ^ Nicholas et al. (2009)

2389C>T and

7781delAG

Algeria 1 family,

3 patients

�4.5 to �6 SD Mild to severe Speech delay Hypoplasia of the

frontal lobes, moderate

posterior parietal atrophy,

anterior orientation of the

insula, thin CC, gyral

simplification

Saadi (2009)

Unable to write,

read and count

at adult age

CENPJ

17delC North 2 families ? ? Bond et al. (2005)

Pakistan

3704A-T Brazil 1 family ? ? Bond et al. (2005)

c.3243–

3246delTCAG

Pakistan 1 family <�3 to 5 SD

(8–13 y)

2 with moderate

mental retardation

(IQ 45–50)

- Unable to read or

write, could not speak

simple phrases and

did not have basic

self-care skills

- No malformations - Normal standard

lymphocyte

karyotype

Gul et al. (2006)

1 severe mental

retardation

(IQ 30–35)

- No other neurological

problems

- EEG normal

STIL

3655 delG India 3 families <�5 to 13 SD

(5–23 y)

- Mild to severe - Delayed developmental

milestones, speech delay

Kumar et al. (2009)

3715C>T - ‘‘No history of any

neurological deficits or

any malformations’’

IVS16 + 1G>A

All mutations are homozygous unless noted. (&) �5 to �11 SD for all patients from Bond et al. (2003); (#) �4 to �9 SD for all patients from Gul et al. (2006); (*) 5 families for both 3978G>A and 9557C>G from Gul; (§) Social

Quotient; (^) OFC<�3 SD for all patients from Nicholas et al. (2009); (�) heterozygous mutation carriers with small heads (OFC �2 to �3.5 SD).* Patients (n = 2) with primary microcephaly due to MCPH1 mutations with PPC syndrome (microcephaly, short stature and Premature Chromosome Condensation): for the first one, height was about �1 SD, for the second one,

between�4 and �5 SD. In PCC syndrome, Trimborn et collaborators noticed that gyral simplification was more marked (resembling pachygyri) than in MCPH1 related patients without PPC phenotype and periventricular neuronal

heterotopias were also present.

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Fig. 3. Position of mutations within the MCPH genes MCPH1, CDK5RAP2, ASPM, CENPJ and STIL. The coding and non-coding regions of the following MCPH genes are drawn to scale,

and the exons are depicted as boxes (reference sequences according to NCBI genome viewer build 35.1 and Ensembl release 54): (A) Microcephalin, MCPH1; (B) CDK5RAP2, cyclin-

dependent kinase 5 regulatory associated protein 2; (C) ASPM, Abnormal spindle-like, microcephaly associated; (D) CENPJ, Centromeric protein J; (E) SIL, SCL/TAL1 interrupting

locus. The localization and type of published mutations are given as well as the number of times a specific mutation has been reported. For cDNA sequence, the reference sequence.

KnownandpredictedMCPHproteindomainsarepresented,andthefull lengthproteinsaregivenwhenseveralisoformsexist.ForCDK5RAP2,theexactpositionsofthetworeported

structuralmaintenanceofchromosome (SMC)domains(Evans etal.,2006)withintheprotein(SMCAA137–499,SMC_NAA793–1040;accessionno.CAI16963)aswell asthatofthe

interaction site with CDK5R1 (AA1726–1768) are not clear and can only be predicted. For the CENPJ gene, Tang and coworkers reported a PN2-3 domain (AA311–422), which

containsa microtubuledestabilizing motif,and a microtubule bindingdomain(AA423–607) (Cormier etal.,2009).For theSTIL gene, a PIN1-bindingdomainhas beenreported tobe

localized at AA567–704 (Campaner et al., 2005), but its location is given as AA583–759 in Genecard and AA584–779 in Uniprot. This site is not cited in the NCBI database.


6. Cyclin-dependent kinase 5, regulatory associated protein 2(syn. CDK5RAP2, C48, CEP215, KIAA1633, MCPH3)

The human cyclin-dependent kinase 5, regulatory associatedprotein 2 gene encodes CDK5RAP2, also referred to as centrosome

associated protein 215 (CEP215) or CDK5 activator binding proteinC48 (C48). Four isoforms have been reported so far. The human fulllength, 1893 amino acid CDK5RAP2 protein contains an N-terminalinteraction site with the gamma-tubulin ring complex (gTuRC),a C-terminal interaction site with CDK5 regulatory subunit 1

Fig. 3. (Continued ).


(CDK5R1, p35, Cdk5 activator binding protein) interacting domainand several coiled-coil domains (Fig. 3B). Ortholog genes withsimilar domain structures are also found in other model organismssuch as the mouse and D. melanogaster. Loss of function causesMCPH3 in humans (MIM*608,201; Bond et al., 2005).

CDK5RAP2 is a centrosome-associated protein, and its mRNA iswidely expressed in human and in embryonal mouse tissue (Bondet al., 2005; Ching et al., 2000). In murine embryos at E15.5, highestlevels were detected in the central nervous system (CNS; Bond etal., 2005). CDK5RAP2 has been associated with centrosomefunction as it shows centrosomal localization in HeLa cellsthroughout the cell cycle, is required for docking of the gTuRC

to the centrosome via its N-terminal domain (but not for gTuRCassembly), and centrosome malfunction occurs in centrosominmutant Drosophila embryos (cnn�/�, CDK5RAP2 homolog gene;Table 4; Moynihan et al., 2000; Paramasivam et al., 2007; Fong etal., 2008; Lucas and Raff, 2007). Specifically, in cnn�/� flies, a loss ofconnection between centrioles and the pericentriolar matrix (PCM)has been reported leading to centrosomes rocketing within thecells (Lucas and Raff, 2007). Amazingly, this was associated withonly subtle defects of asymmetric divisions and no reduction ofbrain size (Lucas and Raff, 2007). CDK5RAP2 is further required forcentrosome cohesion (centrosome duplicates during the cell cyclebut functions as a single microtubule-organizing center until

Fig. 3. (Continued ).


shortly before mitosis; Graser et al., 2007). Moreover, CDK5RAP2interacts with pericentrin, as depletion of pericentrin leads toalmost complete loss of CDK5RAP2 from centrosomes in U2OS cells(Graser et al., 2007).

It has been recently reported that CDK5RAP2 is required forspindle checkpoint regulation, as loss of function leads tochromosome mis-segregation and reduced expression of thespindle checkpoint proteins BUBR1 (budding uninhibited bybenzimidazoles 1 homolog beta) and MAD2 (mitotic arrest-deficient 2) via interaction with their promoters and transcriptionregulation in HeLa cells (Zhang et al., 2009). Chromosomesegregation is initiated by activation of the spindle checkpointtarget APC (anaphase-promoting complex), which functions as anE3 ligase when bound to its activator CDC20 and drives cells frommetaphase into anaphase by inducing degradation of securin and

Fig. 4. Symmetric and asymmetric cell division. The orientation of the spindle (red li

centrosomes (red triangles). Alignment parallel to the neuroepithelium within progenit

pool. An alignment of the spindle perpendicular to the neuroepithelium results in asymm

committed precursors such as neuronal committed precursors. (For interpretation of the

the article.)

mitotic cyclins (Zhang et al., 2009). The spindle checkpoint ensuresthat the CDC20-dependent activation of APC does not occur untilall chromosomes have achieved bipolar kinetochore-microtubuleattachment (Zhang et al., 2009). The spindle checkpoint proteinsBUBR1 and MAD2 are part of an inhibitory complex for APC bysequestering CDC20 through directly binding and thus inhibitingAPC activity (Zhang et al., 2009). Zhang and coworkers suggested amodel in which down-regulation of CDK5RAP2 reduces its bindingto CDC20 and inhibits transcription of BUBR1 and MAD2,consequently decreases BUBR1 and MAD2 protein levels andreleases BUBR1-and MAD2-sequestered CDC20. Thereby, morefree CDC20 would be available to bind chromatin, to activate theAPC and to thus allow transition from metaphase to anaphase inthe presence of unattached kinetochores and/or kinetochores thatlack tension in CDK5RAP2-inhibited cells.

ne) to the neuroepithelium (yellow line) is dependent on the positioning of the

or cells results in symmetrical divisions and thereby an increase of the progenitor

etric cell divisions which maintain the progenitor pool cell numbers while producing

references to color in this figure legend, the reader is referred to the web version of

Fig. 5. Schematic diagram of processes in which MCPH proteins are involved. MCPH proteins play a role in physiological brain development, response to damage and cell cycle

regulation. (A) Centrosomees (yellow dots) attach mitotic spindles (brown lines) of microtubules attached to condensed chromosomes (blue lines) during cell division. The

orientation of the spindle to the neuroepithelium is dependant on the positioning of the centrosomes. Alignment parallel to the neuroepithelium within progenitor cells results

in symmetrical divisions and thereby an increase of the progenitor pool. An alignment of the spindle perpendicular to the neuroepithelium results in asymmetric cell divisions

which maintain the progenitor pool cell numbers while producing committed precursors such as neuronal committed precursors. Progenitors give rise to various cell types such

as neurons and glial cells (astrocytes, oligodendrocytes, microglia) that further differentiate/mature and migrate. The cortical size depends not only on the generation,

maturation and migration of cells, but also on the survival of generated cells. (B) Various insults such as ionizing radiation, chemicals and free radicals can induce DNA damage

and thereby initiate cancer if this damage is not repaired. (C) The cell cycle is required for cell growth and cell division into two daughter cells; to achieve this, cells must perform

DNA synthesis and mitosis. The S-phase stands for synthesis and replicated the genome. The G2 phase is GAP-2 and needed for cell growth and preparation for mitosis. The last

phase is M and stands for mitosis in which cells segregate duplicated chromosomes. Cell cycle regulation is important for proliferation/differentiation of cells and for cell death.

Proper progression through the cell cycle is monitored by checkpoints that sense possible defects during DNA synthesis and chromosome segregation. Activation of these

checkpoints induces cell cycle arrest through modulation of cyclin-dependent kinase (CDK) activity. Cell cycle arrest allows cells to properly repair these defects, thus preventing

their transmission to the resulting daughter cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6. Localization of MCPH proteins in dividing cell. MCPH proteins are highly

localized to the centrosome except for STIL which is localized in the pericentriolar

region. Abbreviations: Microcephalin, MCPH1; CDK5RAP2, cyclin-dependent kinase

5 regulatory associated protein 2; ASPM, abnormal spindle-like, microcephaly

associated; CENPJ, centromeric protein J; SIL, SCL/TAL1 interrupting locus.


CDK5RAP2 inhibits indirectly CDK5 (via inhibition of CDKR1)(Ching et al., 2000; Wang et al., 2000), a highly conserved serinethreonine kinase present most abundantly in postmitotic neuronsof the CNS (for review of CDK5 function see Dhariwala andRajadhyaksha, 2008; Zhang and Herrup, 2008). The nuclearlocalization of CDK5 allows neurons to remain postmitotic in vitro

(Zhang et al., 2008). CDK5 is otherwise an unusual member of theCDK protein family since, unlike other CDKs, it is not believed toregulate a typical cell cycle. CDK5 is pivotal for neuronal migrationand is implicated in processes such as synaptic transmission andneurodegeneration (Dhariwala and Rajadhyaksha, 2008; Dhavanand Tsai, 2001). Cdk5 knockout mice (Cdk5�/�) display perinatalmortality, neuronal migration defects, neurodegeneration (possi-bly via a neurofilament transport defect), but apparently no changeof brain size (Ohshima et al., 1996). Similarly, Cdk5r1 knockoutmice (Cdk5r1�/�) display sporadic adult lethality, sporadicseizures, neuronal migration defects, but no reported change ofbrain size (Chae et al., 1997; Gupta et al., 2003; Kwon and Tsai,1998; Pareek et al., 2006).

7. Abnormal spindle-like, microcephaly-associated (ASPM,MCPH5)

The human abnormal spindle-like, microcephaly-associatedprotein gene encodes ASPM, also referred to as abnormal spindle-like homolog. The human full length, 3477 amino acid ASPM

Table 4MCPH animal models overview.

MCPH (human gene) Animal (gene) Animal model phenotype References

MCPH1 (MCPH1) mcph1�/� - Uncoordinated centrosome and nuclear cycles Brunk et al. (2007), Rickmyre et al. (2007)

- Slowed mitotic entry, premature chromosome

condensation, mitotic entry with unreplicated DNA,

genomic instability, premature separation and

centrosome detachment

- Chk2-mediated mitotic arrest

- Adult brains are reportedly of normal size but have

defects in mushroom body structure

MCPH3 (CDK5RAP2) cnn�/� - Centrosome dysfunction (centrosome ‘rocketing’ within

the cells due to loss of connection between

centrosomes and pericentriolar matrix)

Lucas and Raff (2007)

- Only subtle defects of asymmetric divisions

- Brains are reportedly of normal size

MCPH5 (ASPM) asp�/� - Mitotic arrest in flies Casal et al. (1990)

- Centrosome do not function correctly as

microtubule-organizing center

- Mitotic spindle positioning defect

- Cell cleavage plane positioning defect during asymmetric

cell division

- Spermatogenesis defect

MCPH6 (CENPJ) dsas4�/� - Centrioles are lost during embryonic development Basto et al. (2006), Stevens et al. (2007)

- Slow mitotic spindle assembly

- Abnormal asymmetric divisions of larval neuroblasts

- Mutant flies develop into morphologically normal adults

without cilia or flagella and die shortly after birth

MCPH7 (STIL) - Embryonal lethal (E10.5) Pfaff et al. (2007), Izraeli et al. (1999)

Sil�/� - Developmental abnormalities appear around E7.5–8.5

- Reduced size, limited developmental progress, midline

neural tube defects, abnormal left–right development

- Increased rates of apoptosis

- Decreased rates of proliferation

- Abnormal expression of lefty2, nodal, Pitx2, Patched,

Gli1, Shh, Hnf3b

- Embyryonal lethal

Sil�/� - Disorganized mitotic spindles

- Lack one or both centrosomes

- Increased rates of mitosis

cnn, Centrosomin; dsas, drosophila spindle assembly abnormal. ( ) Drosophila; ( ) mouse; ( ) zebra fish.


protein contains two calponin homology (CH) domains and severalputative calmodulin-binding IQ motifs (Fig. 3C). Orthologgenes with similar domain structures are also found in othermodel organisms. Loss of function causes MCPH5 in humans(MIM*605,481; Bond et al., 2002; Kumar et al., 2002).

ASPM plays a role in mitotic spindle function includingorientation of cleavage plane. The spindle apparatus dictates theplane of cell cleavage, which is critical in the choice betweensymmetric or asymmetric division. Spindle positioning is con-trolled by an evolutionarily conserved pathway. ASPM localizes tothe centrosome in the interphase and to mitotic spindle poles, fromprophase through telophase, in murine embryonic neuroepithelial(NE) cells and primary stem cells as well as progenitor cells of themammalian brain (Zhong et al., 2005; Fish et al., 2006). ASPMmaintains symmetrical cell divisions and is down-regulated withthe switch from proliferative to neurogenic divisions (Fish et al.,2006). Knock-down of Aspm in telencephalic NE cells by RNAinterference induced a lack of Aspm protein at mitotic spindlepoles and an alteration of the cleavage plane (orientation lessfrequently perpendicular to the ventricular surface of theneuroepithelium) (Fish et al., 2006). The authors reported thatthe latter alteration in the cleavage plane orientation increased theprobability that the cells underwent asymmetric division andconcomitantly decreased the number of neuroepithelial progenitorcells since a large proportion of neuroepithelial cell progeny wasfound in the neuronal layer. Recently, it has been reported that

ASPM promotes meiotic spindle organization/positioning viaaccumulation of LIN5 at meiotic and mitotic spindle poles (vander Voet et al., 2009); spindle rotation during maternal meiosisrequires LIN5, ASPM, calmodulin CMD1, and dynein. The authorsargue that the LIN5 complex LIN5/ASPM/CMD� at spindle poles isimportant for dynein function and thus cleavage plane orientationduring mitotic cell divisions (van der Voet et al., 2009). ASPM,moreover, localizes at midbodies and has a possible role in cellularabscission (Paramasivam et al., 2007).

The study of mutant Drosophila (asp�/�) revealed that ASPMmight not only be important for mitotic spindle positioning andfor correct positioning of cell cleavage plane during asymmetriccell division, but also for centrosome function as a microtubule-organizing center ((MTOC) Table 4). Asp gene mutations causemitotic arrest in flies, and Drosophila males display defectsduring spermatogenesis: (i) variable size of nuclei and neben-kerns of early spermatids, which are also multinucleate insteadof having single and uniformly sized nuclei; (ii) elongatingspermatids in which abnormal-sized mitochondrial derivativeselongate alongside more than one axoneme; (iii) failures in theindividualization process, where abnormal spermatids remainsyncytial, and seem to be eliminated during the coiling stage(Casal et al., 1990).

ASPM is down-regulated post ionizing radiation (Fujimori et al.,2008), and Aspm down-regulation decreases BRCA1 (Zhong et al.,2005). ASPM mRNA is over-expressed in transformed human cell


lines and tumors, and its increased expression is positivelyassociated with proliferation of glioblastoma cells (Hagemannet al., 2008).

8. Centromeric protein J (syn. CENPJ, CPAP, LAP, LIP1, MCPH6)

The human centromeric protein J gene encodes CENPJ, alsoreferred to as centrosomal P4.1-associated protein (CPAP), LAG-3-associated protein (LAP) or LYST-interacting protein 1 (LIP1). Thehuman full length, 1338 amino acid CENPJ protein contains a 112-amino acid long microtubule destabilizing motif PN2-3 (Hung etal., 2004) and two C-terminal 14-3-3 binding sites (Chen et al.,2006) (Fig. 3D). Ortholog genes with similar domain structures arealso found in other model organisms. Loss of function causesMCPH6 in humans (MIM*609,279).

CENPJ plays a role in centrosome and spindle function. Theprotein shows centrosomal localization throughout the cell cyclein a microtubule-independent way and is associated to thegamma-tubulin complex (Hung et al., 2000). It has been suggestedthat CENPJ regulated microtubule dynamics at the centrosome,and such a precise regulation of microtubule assembly anddisassembly at kinetochores and centrosomes is thought to beimportant for the maintenance of the spindle structure andchromosome segregation during mitosis (Hung et al., 2004).Expression of a ‘destabilizing domain’ (residues 311–422) was ableto destabilize microtubule nucleation in HeLa cells (Hung et al.,2004). This domain directly recognized the plus end of micro-tubules and inhibited microtubule nucleation from the centro-some, and it also bound to tubulin dimers, resulting in thedestabilization of microtubules (Hung et al., 2004). Over-expres-sion of this CENPJ domain inhibited cell proliferation and inducedapoptosis in HeLa cells after G2/M arrest (Hung et al., 2004).

CENPJ interacts with the N-terminal domain of the p65 subunit(RelA) of NF-kappaB, a transcription factor important for variouscellular events such as inflammation, immune response, prolifera-tion and apoptosis, in 293T (immortalized human embryonickidney HEK) and MCF-7 (human breast adenocarcinoma) cell lines(Koyanagi et al., 2005). It has been suggested that CENPJ functionsas a co-activator of NF-kappaB-mediated transcription (likely viap300/CREB-binding protein) as (i) CENPJ over-expression in theabove named cell lines enhanced NF-kappaB-dependent transcrip-tion induced by tumor necrosis factor-alpha (TNFalpha), (ii) CENPJdown-regulation via siRNA resulted in decreased activation of NF-kappaB by TNFalpha, and (iii) CENPJ interacts with the co-activatorp300/CREB-binding protein (Koyanagi et al., 2005). CENPJ deple-tion via siRNA further lead to an increase of multiple spindle poles,mitosis arrest and apoptosis. Direct interaction between CENPJ and14-3-3 in a cell cycle-dependant manner (significant reductionwhen cells enter mitosis) has been demonstrated in293T andMCF-7 cells (Chen et al., 2006). While the most common mode of14-3-3 function is the sequestration of its target proteins into acompartment such as the cytoplasm to modify their functions, adisruption of the 14-3-3 binding site did not interfere with CENPJtargeting the centrosome (Chen et al., 2006). Since CENPJ has beenreported to translocate to the nucleus after extracellular stimula-tion for example with TNFalpha, it needs to be further analyzedwhether an association with 14-3-3 leads to such a translocation(Chen et al., 2006). Further, the association between CENPJ and 14-3-3 may be important for cell cycle control since 14-3-3 proteinsplay a role in cell cycle control (Chen et al., 2006; Hermeking,2003).

Sas-4�/� mutant Drosophila (spindle assembly abnormal Sas isCENPJ homolog) start to lose their centrioles during embryonicdevelopment until, by third-instar larval stages, no centrioles orcentrosomes are detectable (Basto et al., 2006; Stevens et al., 2007;Table 4). Mitotic spindle assembly is slow in mutant cells, and

approximately 30% of the asymmetric divisions of larval neuro-blasts are abnormal. Nevertheless, mutant flies develop with nearnormal timing into morphologically normal adults. These flies,however, have no cilia or flagella and die shortly after birth becausetheir sensory neurons lack cilia. Thus, centrioles are essential forthe formation of centrosomes, cilia and flagella, but, remarkably,they are not essential for most aspects of Drosophila development.

In C. elegans, sas-4 is a centriolar protein that controlscentrosome size (centrosomal organization) (Kirkham et al.,2003). Centrosomes consist of a centriole pair surrounded bypericentriolar materia, and it is acknowledged that centrioles arerequired to organize PCM to form a structurally stable organelle.Centriole duplication fails in the absence of sas-4, and partialdepletion of sas-4 results in structurally defective centrioles thatcontain reduced levels of sas-4 and organize proportionally lesspericentriolar material (stunted centrioles, smaller centrosomes(Kirkham et al., 2003)). The role of sas-4 in centrosome duplicationwas further underlined by FRAP (fluorescence recovery afterphotobleaching) studies demonstrating that sas-4 was recruited tothe centrosome once per cell cycle, at the onset of the organelleduplication cycle in the S phase (Leidel and Gonczy, 2003;Dammermann et al., 2008). Centriolar sas-4 remains in dynamicequilibrium with the cytoplasmic pool until late prophase, when itis stably incorporated in a step that requires gamma-tubulin andmicrotubule assembly (Dammermann et al., 2008). It has beendemonstrated in staged one-cell C. elegans embryos that daughtercentriole assembly begins with the formation and elongation of acentral tube followed by the peripheral assembly of nine singletmicrotubules (Pelletier et al., 2006). The five proteins zyg-1(ZYGote defective, embryonic lethal), sas-4, sas-5, sas-6, and spd-2(spindle defective 2) have been identified as being essential forcentriole formation, and they are recruited in a specific order(Pelletier et al., 2006; Delattre et al., 2006): (i) tube formation andelongation is dependent on the sas-5 and sas-6, and the assemblyof singlet microtubules onto the central tube depends on sas-4; (ii)centriole assembly is triggered by an upstream signal mediated byspd-2 and zyg-1. Knowledge on the pathway of daughter centrioleassembly in C. elegans could have general relevance for centrioleassembly in other organisms.

9. SCL/TAL1-interrupting locus (syn. STIL, SIL, MCPH7)

The human SCL/TAL1-interrupting locus gene encodes STIL, alsoreferred to as SCL-interrupting locus (SIL). STIL is an immediateearly gene that encodes a cytosolic protein of 150 kDa whosefunction is not fully understood and which lacks homology to anyknown protein families or motifs. Two isoforms have beenreported. The human full length 1287 amino acid protein containsa putative nuclear localization signal and a C-terminal domainsimilar to that of TGF-beta (Karkera et al., 2002) (Fig. 3E). Orthologgenes with similar domain structures are also found in other modelorganisms such as the mouse and D. melanogaster. Three mutationsof the STIL gene leading to a truncated gene product in five Indianfamilies with MCPH have been reported (Kumar et al., 2009).

STIL has been in the focus of cancer research for decades sincegenomic rearrangements containing the STIL gene have beenassociated with T-cell acute lymphoblastic leukemia (ALL) (Aplanet al., 1990, 1992; Brown et al., 1990). In these tumor cells,deletions of the STIL gene lead to a juxtaposition of 50 element(promoter) to the downstream SCL gene and thus resulted in aninappropriate SCL mRNA expression regulated by the STIL

promoter (Aplan et al., 1992). Moreover, an increased expressionof STIL in multiple cancers that correlated with the expression ofmitotic spindle checkpoint genes and with increased metastaticpotential (Erez et al., 2004; Ramaswamy et al., 2003). Only


recently, it has been reported that homozygous mutations of theSTIL gene cause MCPH in humans (Kumar et al., 2009).

STIL is expressed throughout the cytosol with increasedexpression in the perinuclear region that likely plays a role inmitotic entry (cell cycle progression G2-M), apoptosis control andcentrosome function. In HeLa and HEK293T cells, endogenous STILwas detected at the poles of the mitotic spindle in the metaphase(where the microtubules coalesce adjacent to the centrosome),while cells in the anaphase did not show this localization andinterphase cells expressed almost no STIL. STIL is phosphorylatedin mitosis following the mitotic spindle checkpoint. Phosphory-lated STIL interacts with PIN1 (peptidyl prolyl isomerase) which inturn regulates a subset of mitotic phospoproteins (Campaner et al.,2005). STIL expression is regulated by the transcription factor E2F,and it is required for E2F induced transition through mitosis (Erezet al., 2008). The protein is expressed in proliferating cells and isdown-regulated when cellular proliferation ceases because ofserum starvation, contact inhibition or induction of terminaldifferentiation (Izraeli et al., 1997). STIL is essential for mitoticentry and survival of cancer cells (Erez et al., 2007). It influencescancer cell proliferation via de-repression of GLI1 (glioma-associated oncogene homolog) from Suppressor of Fused (SUFU)(Kasai et al., 2008). Moreover, knock-down of STIL in cancer cellscaused a mitotic arrest through a severely delayed entrance intomitosis (G2 to M phase transition), decreased the activation of theCDK1 (CDC2)-cyclin B complex and induced apoptosis in a p53-independent manner (Erez et al., 2007).

Data obtained from zebrafish and HeLa cells indicate that STILmay not only play a role in centrosome function/duplication, butalso in organizing the mitotic spindle. Zebrafish Danio rerio

cassiopeia mutants (loss-of-function mutation in zebrafish sil)have an embryonic lethal defect (Pfaff et al., 2007). They showincreased rates of mitosis, as detected by phospho-H3 immunos-taining, but mitotic cells have extremely disorganized mitoticspindles and often lack one or both centrosomes. In accordancewith these findings, knock-down of STIL by short hairpin RNA(shRNA) in HeLa cells resulted in dividing cells with disorganizedmitotic spindles.

Sil mRNA is expressed in many mouse tissues with highestlevels in bone marrow, thymus, spleen, colon and stomach(Collazo-Garcia et al., 1995). Homozygous sil knockout mice(Sil�/�) display developmental abnormalities around embryonalday (E) 7.5–8.5 and die after E10.5 (Izraeli et al., 1999) (Table 4). Inaddition to reduced size and limited developmental progresscompared to wild-type embryos, Sil mutants have prominentmidline neural tube defects including a delay/failure of neural tubeclosure and holoprosencephaly, abnormal left–right development(randomized heart looping direction), abnormal expression ofLefty2 (left–right determination factor 2), Nodal, Pitx2 (paired-likehomeodomain transcription factor 2), Patched, Gli1, Shh (sonichedgehog), and Hnf3b (hepatocyte nuclear factor 3-beta, foreheadbox A2), increased rates of apoptosis and decreased rates ofproliferation. These latter findings, the reduced expression of genesdownstream of sonic hedgehog (Shh), indicate that STIL may bepart of the Shh pathway. Indeed double mutants of STIL andPatched is consistent with the hypothesis that STIL functionsdownstream of Patched in the Shh pathway (Izraeli et al., 2001).

10. MCPH and cerebellum

Developmentally controlled proliferation, differentiation, mi-gration and apoptosis of cells through processes such ascentrosome function, cell cycle control, spindle assembly anddivision plane orientation are not unique for the cerebral cortex.They also occur throughout the development of the cerebellarcortex. Why a loss of MCPH protein function does not lead to a

gross reduction of the cortex in the cerebellum as it does in thecerebrum is unknown and raises questions regarding the differ-ence in cortex development within the brain. So far, little attentionhas been paid to measuring exactly the size of the cerebellar cortexin patients with MCPH. When doing this, we did detectedcerebellar atrophy in individual patients (unpublished data).Moreover, the role of MCPH proteins have not been studied incerebellar development, and it is, thus, not known whether MCPH

genes are expressed in this context.Defects in other, non-MCPH genes also result in primary

autosomal recessive microcephaly. In a large consanguineousMoroccan family with marked prenatal-onset microcephaly,mutations in the T-box transcription factor gene EOMES (homologof Xenopus Eomesodermin) were detected in affected individuals.In addition to primary microcephaly (Baala et al., 2007), theseindividuals also showed corpus callosum agenesis, bilateralpolymicrogyria, ventricular dilatation, disproportionate pons andcerebellar hypoplasia (Baala et al., 2007). Further, mutations in thecalcium/calmodulin-dependent serine protein kinase gene CASK

have been reported in patients with mental retardation, primarymicrocephaly and hypoplasia of the brainstem and cerebellum(Najm et al., 2008). CASK, a member of the membrane associatedguanylate kinase family, is a multi-domain scaffolding protein thatinteracts with the transcription factor Tbr1 (T-box brain 1) andother genes involved in cortical development such as reelin (RELN).It targets to neuronal synapses and regulates trafficking, targetingand signaling of ion channels (Najm et al., 2008). It will beintriguing to decipher the interaction of these proteins with thoseassociated with MCPH.

11. The role of MCPH proteins in the evolution of the cerebralcortex

The rapid increase in brain size over the past 3–4 million years,and the associated increase in cognitive skills is one of the moststriking features of hominid evolution. MCPH genes are obviouscandidates for this strong evolutionary movement. Early studies ofthe ASPM sequence in great apes show that its evolution issignificantly accelerated, especially in the lineage from the lasthuman/chimpanzee ancestor to humans shows an excess of non-synonymous over synonymous substitutions, a characteristic ofpositive selection. On average, ASPM fixed one mutation every300,000–400,000 years since the human lineage diverged fromchimpanzees some 5–6 million years ago (Evans et al., 2004b). Onegenetic variant of ASPM in humans arose about 5800 years ago andhas since reached an allelic frequency of about 30% (Mekel-Bobrovet al., 2005). Similarly, in MCPH1, a recent variation, whichoccurred 37,000 years ago spread in human population morerapidly than what can be explained by simple genetic drift in asmall population, to reach a frequency of about 70% (Evans et al.,2006, 2005). A similar phenomenon has been observed with thetwo other MCPH genes.

The role of ASPM in the evolution of brain size and cognitivefunction is, however, not without controversy. Studies haveaddressed whether the changes in brain size across the clade ofprimates are correlated to the molecular evolution of the ASPMgene. Two large exons 3 and 18 of ASPM for 23 primate specieswere sequenced. These exons were chosen because they accountfor 70% of the ASPM protein and most of the mutations that occurin microcephaly are found in these exons. Results showed thatpositive selection in ASPM is significantly correlated with cerebralcortex size and not with whole brain size or cerebellum size. This isconsistent with the expression report of ASPM which shows thatASPM expression is limited to the cerebral cortex. This studysuggests that different parts of the brain may have differentevolutionary and functional differentiation based on their distinct


genetic basis. This is in contrast with several studies which havetried to correlate ASPM genotype with brain size variation inhumans (Evans et al., 2004a; Ali and Meier, 2008). Similar analysishas also shown that there have been 45 variations in themicrocephalin gene positively selected within the last 25–30million years of evolution from simian ancestors to modernhumans.

Obviously, the central question is which physiological parame-ter is affected by the Darwinian selection process. The functionalrole of the adaptive evolution of the MCPH genes remainsspeculative, and no clear answer has emerged from geneticepidemiology. The motor of positive selection of the new alleleshas been reckoned to be increased cognitive power. A largeassociation study between the two most recent polymorphismsand IQ has been applied to five replicate samples from fiveindependent populations with a total of about 2400 individuals.The authors concluded that intelligence, as measured by these IQtests, was not detectably associated with the presence of the mostrecent allele of either the ASPM or the Microcephalin gene. In anassociation study of OFC and SNPs located in the ASPM and MCPH1

genes, the non-synonymous SNP rs1057090 (V761A) located in theBRCT domain of the MCPH1 gene is significantly associated with anincreased cranial volume in a Chinese population of 867 unrelatedmales. No recent selection signal could be detected regarding thisSNP, leading the authors to suggest that brain volume variation inhuman populations is neutral or under very weak selection inrecent human history. These results (Wang, 2008) remain to beaffirmed. From such an evolutionary perspective, primary micro-cephaly can be viewed as an example of ‘evolutionary retrogres-sion’ (or an atavistic mutation) whereby the brain dimensions ofthe affected individuals revert back to the level resembling that ofvery early hominids (Evans et al., 2004b; Wang and Su, 2004), aprobably simplistic interpretation as hominids had small brains,but also functional ASPM. However, other studies have come to adifferent conclusion.

It has been noted by Bond et al. that the predicted ASPMproteins encode systematically larger numbers of repeated ‘IQ’domains between flies, mice, and humans, with the predominantdifference between Aspm and ASPM being a single large insertioncoding for IQ domains. One of the most notable trends inmammalian evolution is the massive increase in size of thecerebral cortex, especially in primates. The authors suggested that,in view of evolutionary considerations, these findings suggest thatbrain size is controlled in part through modulation of mitoticspindle activity in neuronal progenitor cells. Humans withautosomal recessive primary microcephaly show a small butotherwise grossly normal cerebral cortex associated with mild tomoderate mental retardation. The authors suggested that geneslinked to this condition offer potential insights into the develop-ment and evolution of the cerebral cortex. This hypothesis is highlydiscussed with subsequent studies supporting or opposing it(Ponting and Jackson, 2005; Evans et al., 2004b; Mekel-Bobrovet al., 2005; Ali and Meier, 2008; Balter, 2005, 2006a,b; Bond andWoods, 2006; Cotoi et al., 1980; Currat, 2006; Frost, 2008;Kouprina et al., 2004; Ponting, 2006; Richards, 2006; Stern andWoods, 2006; Zhang, 2003).

Interestingly, the expansion of IQ repeats has been observedin other non-primate mammalians (see http://www.uniprot.org/uniprot/?query=domain:%22IQ+domain*%22), and raises thequestion of the physiological process which really benefits fromthe selection pressure. As those mammals have not seen thedramatic brain expansion observed in primates, we canspeculate that positive selection is exerted on another parameterregulating reproductive fitness: even if ASPM function seems tobe redundant in tissues outside brain in fetuses and postnatally,nothing is known on its role in gametogenesis or early

embryonic development. Positive selection pressure could beexerted on a more fundamental process than brain size—and, tobe provocative, increased brain size in the primate taxon couldperhaps be a ‘‘side effect’’ of a selection mechanism that affectsall mammalians.

12. Other proteins that show an overlap with MCPHpathomechanisms and phenotype

Centrosome, cell cycle and/or DNA repair-linked proteins havebeen associated with microcephaly phenotypes close to that ofMCPH. The most striking example is the centrosomal proteinpericentrin (PCNT). Mutations in the PCNT gene cause a clinicalphenotype continuum from MCPH1-reminicient patients to Seckeltype 4 syndrome (Griffith et al., 2008) and microcephalicosteodysplastic primordial dwarfism type II (MOPDII) patients(Rauch et al., 2008). Microcephaly, short stature and mentalretardation are the common points of these three syndromes.Microcephaly is isolated in MCPH1 and more pronounced for intwo other diseases. Short stature has been reported only inindividual MCPH patients while striking growth retardation(Seckel) or dwarfism (MOPDII) is prominent in the other diseases.In these diseases, the facial appearance with sloping forehead,micrognathia, prominent midface and upward slanting of thepalpebral fissures is characteristic. Tibelius and coworkers recentlyshowed that a lack of MCPH1 or PCNT results in a loss of CHK1 fromcentrosomes, using two approaches (patient-derived cells carryingMCPH1 or PCNT mutations and cells treated with MCPH1 and PCNTspecific siRNAs) (Tibelius, 2009). Immunoprecipitation of GFP-MCPH1 using an anti-GFP antibody leads to co-immunoprecipita-tion of endogenous CHK1, suggesting that MCPH1 and CHK1interact with each other and that MCPH1 targets CHK1 to thecentrosome. PCNT immunostaining was diminished in lympho-blastoid cell lines derived from patients with MCPH1 mutationsand in U2OS cells treated with MCPH1 siRNA. Immunoprecipitationof GFP-MCPH1 leads to co-immunoprecipitation of endogenousPCNT. Based on their data, the authors suggest that MCPH1 andPCNT interact with each other. As a further finding that underlinessuch an interaction between PCNT and CHK1, Tibelius andcoworkers demonstrated that PCNT was immunoprecipitated bothfrom control or MCPH1 patient-derived cell lines and thatimmunoprecipitation of endogenous CHK1 lead to co-immuno-precipitation of endogenous PCNT. The authors concluded that (i)PCNT and CHK1 interact with each other independent from thepresence of MCPH1, (ii) MCPH1 contributes to the centrosomallocalization of PCNT and (iii) both MCPH1 and PCNT are required torecruit CHK1 to the centrosome. Microcephaly is also a commonclinical feature in disorders involving DNA repair abnormalitiessuch as ataxia telangiectasia and RAD3-related protein (ATR) genemutation or defective ATR signaling caused Seckel syndrome type1, Fanconi Anemia or Nijmegen syndrome. Future studies devotedto resolving the exact overlap of such gene products known to beinvolved in DNA repair pathways in patients with syndromeswhich include microcephaly as a clinical sign with thosemechanisms implicated in MCPH are needed.

13. Conclusion

Brain size is largely determined by the relative rates ofproliferation and cell death, and proteins encoded by MCPH geneshave been shown to play a prominent role in several key steps ofdevelopmental processes that could be responsible for a markedreduction of the cortical area, such as cell cycle progression, DNA/spindle checkpoints as well as centrosome and spindle apparatusintegrity. Some common pathways have been identified for MCPHproteins (e.g. interaction of MCPH1 and STIL with the transcription

http://www.uniprot.org/uniprot/?query=domain:%22IQ+domain*%22

http://www.uniprot.org/uniprot/?query=domain:%22IQ+domain*%22


factor E2F). Identifying the pathomechanisms underlying MCPHwill not only increase our knowledge of these rare diseases but alsoenhance our understanding of normal brain development and ofevolution of the brain development. Still, many studies have beenperformed in cancer cells in vitro, and murine models are largelylacking. Murine models will be particularly relevant to determinethe precise roles of MCPH proteins in the control of proliferation ofnormal mammalian neural precursor cells as compared to theirrole in lower organisms or cancer cells. Deciphering the molecularpathways associated with MCPH proteins will also allow todetermine the possible interplays between these different MCPHproteins. Future directions for research in the field could also aimat determining the potential involvement of MCPH proteins andrelated pathways in the pathophysiology of microcephalies ofother etiologies including environmentally induced microcepha-lies.

Conflict of interest

None.

Acknowledgements

The authors are members of the m-icronet consortium(Microcephaly-international collaborative research on etiology).Our research is supported by Inserm, Paris 7, Fondation Lejeune,the German Research Foundation (SFB665), PremUp and theFondation pour la Recherche Medicale et Societe Francaise deNeuropediatrie.

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Many roads lead to primary autosomal recessive microcephaly

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