Original article J Med Genet: first published as 10.1136/jmg.2008.062380 on 21 November 2008. Downloaded from The molecular landscape of ASPM mutations in primary microcephaly A K Nicholas,1 E A Swanson,2 J J Cox,1 G Karbani,3 S Malik,3 K Springell,4 D Hampshire,4 M Ahmed,3 J Bond,4 D Di Benedetto,5 M Fichera,5 C Romano,6 W B Dobyns,2 C G Woods1 c Additional tables are ABSTRACT also a diagnosable cause of mental retardation, and published online only at http:// Background: Autosomal recessive primary microcephaly one with a substantial recurrence risk of one in jmg.bmj.com/content/vol46/ (MCPH) is a model disease to study human neurogenesis. issue4 four in subsequent children. In affected individuals the brain grows at a reduced rate 1 The current diagnostic criteria for MCPH are: Department of Medical during fetal life resulting in a small but structurally normal congenital microcephaly more than 23 SD below Genetics, Cambridge Institute for Medical Research, University brain and mental retardation. The condition is genetically age and sex means; mental retardation but no of Cambridge, Cambridge, UK; heterogeneous with mutations in ASPM being most other neurological finding, such as spasticity, 2 University of Chicago, commonly reported. seizures, or progressive cognitive decline; normal Department of Human Genetics, Methods and results: We have examined this further by height and weight, appearance, and results on Chicago, Illinois, USA; studying three cohorts of microcephalic children to extend 6 3 Department of Clinical chromosome analysis and brain scan. Despite this, Genetics, St James’s University both the phenotype and the mutation spectrum. Firstly, in MCPH still remains a clinical diagnosis of exclu- Hospital, Leeds, UK; 4 Section of 99 consecutively ascertained consanguineous families sion. Further, the recurrence risk of the MCPH Ophthalmology and with a strict diagnosis of MCPH, 41 (41%) were phenotype after one affected child (with careful Neuroscience, Leeds Institute of homozygous at the MCPH5 locus and all but two families Molecular Medicine, University elimination of differential diagnoses) in a non- of Leeds, Leeds, UK; had mutations. Thus, 39% of consanguineous MCPH consanguineous family is one in eight, and in a 5 Laboratory of Genetic families had homozygous ASPM mutations. Secondly, in consanguineous family one in six.7–9 Our first aim Diagnosis, I.R.C.C.S. 27 non-consanguineous, predominantly Caucasian families in designing this study was to be able to diagnose Associazione Oasi Maria with a strict diagnosis of MCPH, 11 (40%) had ASPM MCPH with greater accuracy, particularly at an Santissima, Troina (EN), Italy; mutations. Thirdly, in 45 families with a less restricted 6 Unit of Paediatrics and Medical early age, and to examine if the current diagnostic Genetics, I.R.C.C.S. phenotype including microcephaly and mental retardation, criteria accurately reflect the phenotypic spectrum Associazione Oasi Maria but regardless of other neurological features, only 3 (7%) of the disorder. Santissima, Troina (EN), Italy had an ASPM mutation. This report contains 27 novel MCPH can be caused by recessive mutations in mutations and almost doubles the number of MCPH up to seven genes.46 Unexpectedly most of the Correspondence to: associated ASPM mutations known to 57. All but one of Dr C G Woods, Department of known MCPH genes, CDK5RAP2, ASPM and Medical Genetics, Cambridge the mutations lead to the use of a premature termination 10 CENPJ (better known as CPAP ), encode centro- http://jmg.bmj.com/ Institute for Medical Research, codon, 23 were nonsense mutations, 28 deletions or somal proteins, highlighting the importance of the University of Cambridge, insertions, 5 splicing, and 1 was a translocation. Cambridge, CB2 0XY, UK; centrosome in neurogenesis.3 11–13 Despite this, a Seventeen of the 57 mutations were recurrent. There [email protected] common mechanism explaining the role of the were no definitive missense mutations found nor was MCPH genes in neurogenesis has yet to emerge. All there any mutation/phenotype correlation. ASPM muta- Received 5 August 2008 four known MCPH proteins are also present in the Revised 8 October 2008 tions were found in all ethnic groups studied. Accepted 16 October 2008 Conclusion: This study confirms that mutations in ASPM midbody (the microtubular structure linking Published Online First are the most common cause of MCPH, that ASPM daughter cells at the final stage of cytokinesis) on September 30, 2021 by guest. Protected copyright. 21 November 2008 mutations are restricted to individuals with an MCPH and have apparently diverse roles: microcephalin in phenotype, and that ASPM testing in primary micro- DNA repair and chromosome condensation, cephaly is clinically useful. CDK5RAP2 and CENPJ in centriole/centrosome replication, and ASPM in modulating the plane of cytokinesis in neural precursors.14–19 Mutations in Our most defining feature as a species is our brain the ASPM gene at the MCPH5 locus on chromo- with its large size and cognitive functions leading some 1q31.3 have been considered the most 62021 to our great adaptability.1 Many genes are involved common cause of MCPH. Our second aim in in the growth of the developing human brain but this study was to determine the frequency of the identification of those which have a non- ASPM mutations in microcephalic individuals, redundant and crucial role has proved difficult. delineate the spectrum of mutations seen in the Autosomal recessive primary microcephaly ASPM gene, and evaluate the utility of sequencing (MCPH) has emerged as a model disorder in which this gene in clinical practice. to seek such genes, as it is a condition where fetal The primary feature of MCPH is microcephaly— brain growth is significantly reduced (as is head a reduced occipitofrontal head circumference size throughout life), brain architecture is normal, (OFC)—which is an imperfect indicator of micro- and there are no apparent abnormalities in other encephaly (a small brain). Ideally volumetric body systems.2–4 Therefore the genes that cause magnetic resonance imaging (MRI) studies should This paper is freely available online under the BMJ Journals MCPH may be expected to have an important, be performed to quantitate brain volume, but this unlocked scheme, see http:// noticeable and non-redundant role in neurogenesis, is not available in current clinical practice, whereas jmg.bmj.com/info/unlocked.dtl but not other developmental processes.5 MCPH is head circumference charts are tried, tested and J Med Genet 2009;46:249–253. doi:10.1136/jmg.2008.062380 249 Original article J Med Genet: first published as 10.1136/jmg.2008.062380 on 21 November 2008. Downloaded from useful.22–24 The choice of a cut-off value for microcephaly is mutations found were shown to segregate faithfully in the important but arbitrary. Half of mentally retarded individuals family, although the degree of intrafamilial microcephaly varied, are microcephalic725and half of children with OFC of 22SDor as previously reported.6 less have normal intelligence.26 To define microcephaly some authors use an OFC ,2 SD below the mean for age and sex, RESULTS which results in 2% of the general population being considered microcephalic.27 28 Others use ,3 SD so that 0.1% of the general Of the consanguineous families in cohort 1, homozygous ASPM population are microcephalic and most do have mental mutations were found in 39/41 of families (95% of families that retardation. 72429 Added to this are probable ethnic variations were homozygous at the MCPH5 locus, 39% of all consangui- and differences in norms between growth charts. Because of all neous families). Of the families that were homozygous at the of these considerations we use an OFC of more than 23SDto MCPH5 locus with no ASPM mutations, one had a singleton define microcephaly in this study, realising that it could exclude affected child and one had an affected pair of first cousins. In a small number of true microcephalics but by corollary be cohort 2, of non-consanguineous, predominantly Caucasian unlikely to include people with normal brain volumes or individuals, ASPM mutations were found in 11/27 families intelligence. (40%), and in 3/5 sib pairs. In this cohort 19 mutations were found: four were present in other cohorts, all but one led to a premature termination codon and no novel missense mutations METHODS were found. In three families the ASPM mutation was Three cohorts of microcephalic individuals were investigated. homozygous and the remainder were compound heterozygotes. The first cohort (cohort 1) is of 99 consecutively ascertained In cohort 3, of 45 microcephalic individuals with a wider consanguineous families of Pakistani or Arab origin seen by one phenotype we found mutations in three families (7%). One author (CGW), the first 56 of which have been previously partly consanguineous and one non-consanguineous singleton each 6 reported. Seventy-three families had multiple affected members had a homozygous nonsense mutation. Both of these homo- and 26 consisted of a singleton child. No family was more zygous mutations were present in other cohorts. A child from a distantly related than parents being second cousins. All fulfilled third family carried two heterozygous protein truncating the current MCPH diagnostic criteria, with the exception that a mutations. The mutations from all three cohorts and all brain MRI scan was only available in eight families (all showed previously reported mutations are described in table 1 and no architectural anomalies). The second cohort (cohort 2) is of shown in fig 1. 27 non-consanguineous MCPH families of predominantly European origin, including five sib pairs. All had a normal brain scan and conformed to standard MCPH diagnostic criteria. The DISCUSSION third cohort (cohort 3) is of 45 children (from 45 families) that This work brings the total number of different ASPM mutations was examined specifically to answer the questions, ‘‘Are the reported to 57.