HUMAN MUTATION Mutation in Brief #853 (2005) Online

MUTATION IN BRIEF

The First Missense Alteration in the MCPH1 Gene

Causes Autosomal Recessive Microcephaly With an
Extremely Mild Cellular and Clinical Phenotype
Marc Trimborn1,2, Reyk Richter1, Nadine Sternberg1, Ioannis Gavvovidis3, Detlev Schindler3,
Andrew P. Jackson4, Eva-Christina Prott5, Karl Sperling1, Gabriele Gillessen-Kaesbach5, and
Heidemarie Neitzel1*
1
Institut fr Humangenetik, Charit Universittsmedizin Berlin, Germany; 2FU-Berlin, Fachbereich Biologie,
Chemie, Pharmazie, Berlin, Germany; 3 Institut fr Humangenetik, Universitt Wrzburg, Germany; 4MRC Human
Genetics Unit, Western General Hospital Edinburgh,United Kingdom; 5Institut fr Humangenetik,
Universittsklinikum Essen, Germany

*Correspondence to: Prof. Dr. Heidemarie Neitzel, Institute of Human Genetics, Charit -Universittsmedizin
Berlin, Augustenburger Platz 1, 13353 Berlin, Germany; Tel.: +49 30 450566411; Fax: +49 30 450566933; E-
mail: heidemarie.neitzel@charite.de

Grant sponsor: Deutsche Forschunsgemeinschaft DFG; Grant number: NE 531/5-1.

Communicated by Christopher G. Mathew

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder

characterized by mental retardation and congenital microcephaly with a head circumference
at least 4 SD below age and sex means, in the absence of other significant malformations or
neurological deficits. Truncating alterations in the MCPH1 gene have previously been shown
to exhibit a distinct cellular phenotype, with a high proportion of prophase-like cells (>10%)
due to premature chromosome condensation in early G2- and delayed decondensation in
early G1-phase of the cell cycle. We report here the first patient with a homozygous
substitution of a highly conserved threonine residue by an arginine (c.80C>G, Thr27Arg)
localized in the N-terminal BRCT domain of MCPH1. The cellular and clinical phenotype of
this patient is much less pronounced than that of previously described patients with
truncating alterations in the MCPH1 gene. Firstly, the fraction of prophase-like cells
accounts for just 34% of the cell population. Secondly, clinically, he has only a very mild
mental retardation with predominantly delayed motor skills but normal verbal IQ
attainment. Additionally, head circumference was less severely affected, being 2.4 SD at
birth and 3 SD at the age of six years. This justifies reconsideration and widening of the
clinical phenotype definition of MCPH1. 2005 Wiley-Liss, Inc.

KEY WORDS: MCPH1, microcephalin; PCC syndrome; chromosome condensation defect; microcephaly

Received 12 July 2005; accepted 23 August 2005.

2005 WILEY-LISS, INC.

DOI: 10.1002/humu.9382
2 Trimborn et al.

INTRODUCTION
Autosomal recessive primary microcephaly (MCPH) displays genetic heterogeneity with six loci mapped to
date (MCPH1 to MCPH6)(Jackson et al., 1998; Roberts et al., 1999; Jamieson et al., 1999; Jamieson et al., 2000;
Moynihan et al., 2000; Pattison et al., 2000; Leal et al., 2003). Four of the respective genes for these loci have been
identified: MCPH1 encoding microcephalin (Jackson et al., 2002), MCPH3, encoding CDK5RAP2 (Bond et al.,
2005), MCPH5 encoding ASPM (Bond et al., 2002), and MCPH6, encoding CENPJ (Bond et al., 2005). Further
MCPH genes are likely to exist, as 18/56 northern Pakistani families and 5/9 Indian families showed no evidence of
linkage to the known loci (Roberts et al., 2002; Kumar et al., 2004). Clinically, patients with mutations at any of
the loci are indistinguishable. The definitive phenotype for these genes has been proposed to be a pronounced
microcephaly of at least 4 SD below age and sex means with mental retardation in absence of other severe
congenital malformations or significant neurological deficits (Woods et al., 2005).
The hallmark of mutations in the MCPH1 gene (MIM# 606858; MIM# 607117) is a cellular phenotype
detectable in routine cytogenetic preparations in which a high proportion (~10%-20%) of cells have prophase-like
chromosomes. This highly characteristic cellular phenotype is due to premature chromosome condensation in the
early G2 phase of the cell cycle (hence, "premature chromosome condensation" = PCC syndrome) and delayed
decondensation postmitosis. This aberrant regulation thus causes the large numbers of prophase-like cells (PLCs)
that are seen (Neitzel et al., 2002; Trimborn et al., 2004). The MCPH1 gene encodes an 835 amino acid protein,
microcephalin, which contains one N-terminal and two C-terminal BRCT domains (BRCA1 C-terminus). These
domains are present in many cell cycle and DNA repair proteins. To date, only two different truncating mutations
have been reported in MCPH1. Two Pakistani families with an identical haplotype carry the same homozygous
nonsense mutation (c.74G>C; Ser25X) in exon 2 (Jackson et al., 2002). In a Lebanese family a homozygous 1bp
insertion, c.427_428insA, leads to a frameshift resulting in a premature stop codon in exon 6 (p.Thr143AsnfsX5)
(Trimborn et al., 2004).
Here, we report a new patient of Caucasian origin whose cells exhibit the characteristic - although less
pronounced - defect in chromosome condensation. He has a homozygous c.80C>G alteration resulting in a
substitution p.Thr27Arg within the N-terminal BRCT domain of MCPH1.

METHODS

Cytogenetics
Conventional cytogenetic analyses of cultured T-lymphocytes and lymphoblastoid cell lines (LCLs) were
performed using standard techniques. LCLs were established as described elsewhere Neitzel, 1986). For exclusion
of Nijmegen breakage syndrome, the radiosensitivity was determined by analyzing the number of breaks per cell in
50 metaphases per irradiation dose. Irradiation of cells was carried out using the X-ray apparatus Muller MG 150
(UA = 100 kV, I = 10 mA, filter 0.3 mm Ni, dose rate: 2.1 Gy/min) with doses 0.5, 1.0, and 2.0 Gy.

Mutation and SNP analysis

For mutation detection all 14 exons and exonintron boundaries of the MCPH1 gene were PCR amplified
(primers and conditions are available from the authors). The PCR products were cycle sequenced using the Big
Dye Terminator Ready Reaction Mix (Applied Biosystems) and analyzed on ABI 3100 Genetic Analyzer (Applied
Biosystems). Sequences were compared with a reference cDNA sequence (GenBank accession: AX087870).
Primer sequences for the amplification of exon 2 were: MCPH1-Exon2F 5-CTATTGGGCAGGGGATGCTG-3
and MCPH1-Exon2R 5'-CCAATCAGAAGACTGTCATATGAATC-3'. The presence of the mutation was confirmed
by sequencing both strands and by SNP analysis. The site of the mutation of 196 control alleles was analyzed using
the ABI PRISM SNaPshot Multiplex Kit, MCPH1-SNP-Primer: 5-TCCAATGGAACAGAAAATT
ATTCAAAGA-3 and ABI PRISM 3100 GeneScan Analysis Software.
The protein sequences which were deduced from the reference cDNA sequences were analyzed using Blast at
NCBI (hppt://ncbi.nlm.nih.gov/).
 MCPH1 Missense Mutation 3

RESULTS

Clinical data
The proband was referred for diagnostic evaluation at the age of 5 11/12 years because of developmental delay
and microcephaly. He is the first child of a German mother, and his father is unknown. Further family history is
unavailable as he was adopted two weeks after birth. He was born after a Caesarean section at 35 weeks gestation.
Birth weight was 2350 g (-0.7 SD), length 45 cm (-0.5 SD), and head circumference 30,5 cm (-2.4 SD) (Roche et
al.,1987). Early milestones were in the normal range: sitting at 6 months, walking at 12 months and starting to
speak at 18 months. Since the age of 4 years head circumference has not considerably increased. MRI did not
identify any cerebral malformation and EEG was normal. Although his verbal skills are well maintained (verbal IQ
115), he has mild learning difficulties (overall IQ 84) and in particular his fine motor skills are delayed. At the age
of 5 11/12 years his height was 114 cm (-0,7 SD), weight was 18,5 kg (-1,3 SD), and his head circumference 47 cm
(-3 SD). He had upslanting palpebral fissures and a nearly complete bilateral, cutaneous syndactyly of the 2nd and
3rd toes but no other congenital malformations. Smith-Lemli-Opitz syndrome was excluded by biochemical
screening. Thus, cytogenetics analysis was then performed to investigate his microcephaly and developmental
delay and the possibility of Nijmegen breakage syndrome.

Figure 1. Metaphase (A) and prophase-like cell (B) from the patient with the missense alteration c.80C>G. Cellular phenotype
of MCPH1 patients with truncating mutations and the novel missense mutation c.80C>G: proportion of prophase-like cells in
the c.80C>G patient, a patient with the truncating mutation c.427_428insA, a patient with the truncating mutation c.74G>C, and
two normal control subjects in T-lymphocytes after Colcemid treatment for 2 hours and in lymphoblastoid cell lines without
Colcemid treatment (C).
4 Trimborn et al.

Cytogenetic analyses
The chromosome analysis revealed a numerically normal male karyotype. Chromosome breakage analysis after
ionizing irradiation did not show enhanced radiosensitivity and, thus, excluded the diagnosis of Nijmegen breakage
syndrome. However, several attempts to yield high-resolution chromosomes after methotrexate treatment were
unsuccessful with an extremely poor metaphase resolution of 250 bands per haploid genome. Importantly, in
addition to the poor chromosomal resolution (Fig. 1A) we observed an enhanced fraction of prophase-like cells
(PLCs) in the lymphocyte cultures of the patient (Fig. 1B). The percentage of 3.3 % PLCs was clearly less marked
than that seen in patients with truncating mutations in the MCPH1 gene (>10% PLCs), but significantly raised
from that seen in normal controls (0.6 % - 1.5 % prophases) (Fig. 1C). These findings were confirmed by
analyzing cytogenetic preparations of lymphoblastoid cell cultures of the proband, patients with truncating
mutations, and controls (Fig. 1C).

Molecular data
Sequencing of genomic DNA of the patient revealed a homozygous missense mutation c.80C>G in exon 2 of
the MCPH1 gene resulting in the change of the uncharged amino acid threonine to a larger basic arginine residue at
codon 27 (p.Thr27Arg). Parental genotyping was not possible, as the patient had been adopted two weeks after
birth. Microsatellite analysis of the patients DNA revealed heterozygosity for the STRs (D8S504, D8S264,
D8S518) telomeric of MCPH1 and homozygosity for a 19 Mb segment centromeric of MCPH1 (D8S277, D8S503,
D8S560, and D8S1771). These data together with the homozygosity of the missense alteration, indicate likely
consanguinity of the parents rather than a gross deletion of this segment, as this would have been detected by
conventional cytogenetics even at the poor banding resolution of MCPH1 patients.
The c.80C>G alteration was absent from 196 alleles of ethnically matched controls by SNP analysis, indicating
that this is not a common sequence polymorphism. Additionally, multiple sequence alignments of the MCPH1
protein orthologs from diverse species indicate that Thr27 is entirely conserved in all mammalian species and in
the amphibians Xenopus and Ambystoma. In Gallus gallus the uncharged threonine is conservatively substituted
for a neutral alanine (Table 1).

Table 1. Cross species alignment of MCPH1. ClustalW alignment of residues 19-40 of the human microcephalin with the
orthologs of different species. Thr27 is highlighted in black, the amino acids of the partial N-terminal BRCT domain which are
conserved in all vertebrates are highlighted in gray.

Homo sapiens SSNGTENYSKTFTTQLVDMGAK

Great apes SSNGTENYSKTFTTQLVDMGAK
Hylobates lar SSNGTENYSKTFTTQLVDMGAK
Lemur catta SSNGTENYSKTFTNQLVDMGAK
Mus musculus SSKGTENYSRTFAKQLEDMGAT
Rattus norvegicus SSKGTENYSKTFTKQLEDMGAT
Canis familiaris SVNGTENYSKTFTNQLVDMGAK
Felis catus SVNGTENYSKTFTNQLVDMGAK
Bos taurus SANGTENYSKTFRNQLVDMGAK
Xenopus tropicalis SSNRRENYSKTFSQQLVNLGAK
Ambystoma mexicanum SSNRTENYSKTFAQQLLNLGAK
Gallus gallus SSSRTENYSKAFEQQLLDMGAK

DISCUSSION
Virtually all mutations described so far in the four MCPH genes are predicted to result in protein truncation. The
sole exception is the mutation Glu1235Val in a highly conserved Tcp10 binding domain of the CENPJ protein in
MCPH6 (Bond et al., 2005; Hung et al., 2004). So it is significant that we describe here the first missense mutation
in MCPH1. The criteria to predict that the sequence change Thr27Arg is pathogenic and causative for the clinical
MCPH phenotype are based on the following molecular and functional evidence: (1) the mutation is
nonconservative, leading to an alteration in the polarity and charge of the amino acid; (2) Thr27 is highly
conserved in diverse species, despite this being a rapidly, and adaptively evolving gene (Evans et al., 2004;
Ponting and Jackson, 2005) and when altered, only in a conservative manner (Gallus gallus: Thr to Ala); (3) the
alteration is not a common polymorphism, being absent from 196 Caucasian control alleles; and (4) most
 MCPH1 Missense Mutation 5

important, the alteration generates the highly characteristic MCPH1 cellular phenotype of aberrant chromosome
condensation which in combination with the poor banding resolution, appears to be unique to MCPH1
microcephaly and microcephalin gene mutations. In particular, this cellular phenotype has not been described in
any other MCPH deficiency nor in other disorders associated with congenital microcephaly like Nijmegen
breakage syndrome, Ligase IV- and MRE11-deficiency, or Fanconi anemia.
Missense mutations associated with loss of function are important in delineating protein regions with particular
functional relevance. The p.Thr27Arg mutation is located in the conserved region of the N-terminal BRCT domain
of microcephalin, a domain known to be important in cell cycle regulating genes (Huyton et al., 2000). Thus, the
observed functional consequence of mutation in this domain is consistent with the previously identified cell cycle
(chromosome condensation regulation) role identified for this protein (Neitzel et al., 2002; Trimborn et al., 2004),
and we infer that this BRCT domain must be essential for the normal regulation of chromosome condensation.
Overall, the proportion of prophase-like cells with condensed chromatin is considerably lower in cells with
Thr27Arg compared to MCPH1 patients with the two truncating mutations. Consequently, we can not rule out the
possibility that mutations in the MCPH1 gene may result in a very mild cellular phenotype which may not be
easily detectable on routine cytogenetic preparations. In cases with clinical MCPH and poor metaphase resolution,
we therefore suggest that analysis of chromosome preparations without colcemid treatment be performed, since the
application of the spindle poison attenuates the condensation defect by masking the postmitotic effect in early G1.
It is also noteworthy that the patient presented here has a remarkably mild clinical phenotype. The recently
proposed clinical definition/criteria for MCPH (Woods et al., 2005) specifies that head circumference must be at
least 4 SD below age and sex means. In the Thr27Arg patient the head circumference was -2.4 SD at birth and 3,0
SD at the age of 6 years. The mental retardation is also less severe compared to other MCPH patients, with the
patient affected with the Thr27Arg mutation having surprisingly high verbal skills (verbal IQ 115) and an overall
IQ of 84. This current report, therefore provides evidence that mutations in the MCPH1 gene and indeed in other
MCPH genes may well result in a milder phenotype than currently suggested.
As in many other monogenic disorders post gene identification, we have demonstrated unanticipated variability
in the phenotypic expressivity of MCPH1 mutations after identification of the underlying gene. This justifies
reconsideration and widening of the clinical phenotype definition (Woods et al., 2005), which is important not only
for accurate diagnosis and genetic counseling but also to permit detection of further mutations which could
pinpoint specific domains of relevance for chromosome condensation and neurogenesis. In addition, functional
studies will be important to provide further insights into the mechanisms giving rise to the specific clinical and
cellular manifestations of mutations in microcephalin.

ACKNOWLEDGMENTS
The authors thank Sylke Niehage for excellent technical assistance.

REFERENCES
Bond J, Roberts E, Mochida GH, Hampshire DJ, Scott S, Askham JM, Springell K, Mahadevan M, Crow YJ, Markham AF,
Walsh CA, Woods CG. 2002. ASPM is a major determinant of cerebral cortical size. Nat.Genet. 32:316 320.

Bond J, Roberts E, Springell K, Lizarraga S, Scott S, Higgins J, Hampshire DJ, Morrison E, Leal GF, Silva EO, Costa SM,
Karbani G, Rashid Y, Jafri H, Bennett C, Corry P, Walsh CA, Woods CG. 2005. A centrosomal mechanism involving
CDK5RAP2 and CENPJ controls brain size. Nat.Genet. 37:353 355.

Evans PD, Anderson JR, Vallender EJ, Choi SS, Lahn BT. 2004. Reconstructing the evolutionary history of microcephalin, a
gene controlling human brain size. Hum.Mol.Genet. 13:11391145.

Hung LY, Chen HL, Chang CW, Li BR, Tang TK. 2004. Identification of a novel microtubule-destabilizing motif in CPAP that
binds to tubulin heterodimers and inhibits microtubule assembly. Mol.Biol.Cell 15:26972706.

Huyton T, Bates PA, Zhang X, Sternberg MJ, Freemont PS. 2000. The BRCA1 C-terminal domain: structure and function.
Mutat. Res. 460:319332.

Jackson AP, McHale DP, Campbell DA, Jafri H, Rashid Y, Mannan J, Karbani G, Corry P, Levene MI, Mueller RF, Markham
AF, Lench NJ, Woods CG. 1998. Primary autosomal recessive microcephaly (MCPH1) maps to chromosome 8p22-pter.
Am.J.Hum.Genet. 63:541 546.
6 Trimborn et al.

Jackson AP, Eastwood H, Bell SM, Adu J, Toomes C, Carr IM, Roberts E, Hampshire DJ, Crow YJ, Mighell AJ, Karbani G,
Jafri H, Rashid Y, Mueller RF, Markham AF, Woods CG. 2002. Identification of microcephalin, a protein implicated in
determining the size of the human brain. Am.J.Hum.Genet. 71:136 142.

Jamieson CR, Govaerts C, Abramowicz MJ. 1999. Primary autosomal recessive microcephaly: homozygosity mapping of
MCPH4 to chromosome 15. Am.J.Hum.Genet.65:1465 1469.

Jamieson CR, Fryns JP, Jacobs J, Matthijs G, Abramowicz MJ. 2000. Primary autosomal recessive microcephaly: MCPH5
maps to 1q25-q32. Am.J.Hum.Genet. 67:1575 1577.

Kumar A, Blanton SH, Babu M, Markandaya M, Girimaji SC. 2004. Genetic analysis of primary microcephaly in Indian
families: novel ASPM mutations. Clin. Genet. 66:341348.

Leal GF, Roberts E, Silva EO, Costa SMR, Hampshire DJ, Woods CG. 2003. A Brazilian locus for autosomal recessive
primary microcephaly maps to 13q12.2. J.Med.Genet. 40:540 542.

Moynihan L, Jackson AP, Roberts E, Karbani G, Lewis I, Corry P, Turner G, Mueller RF, Lench NJ, Woods CG. 2000. A third
novel locus for primary autosomal recessive microcephaly maps to chromosome 9q34. Am.J.Hum.Genet. 66:724 727.

Neitzel H, Neumann LM, Schindler D, Wirges A, Tonnies H, Trimborn M, Krebsova A, Richter R, Sperling K. 2002.
Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal
recessive condition. Am.J.Hum.Genet. 70:10151022.

Neitzel H. 1986. A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum.Genet. 73:320-
326.

Pattison L, Crow YJ, Deeble VJ, Jackson AP, Jafri H, Rashid Y, Roberts E, Woods CG. 2000. A fifth locus for primary
autosomal recessive microcephaly maps to chromosome 1q31. Am.J.Hum.Genet. 67:1578 1580.

Ponting C, Jackson AP. 2005. Evolution of primary microcephaly genes and the enlargement of primate brains.
Curr.Opin.Genet.Dev. 15:241248.

Roberts E, Jackson AP, Carradice AC, Deeble VJ, Mannan J, Rashid Y, Jafri H, McHale DP, Markham AF, Lench NJ, Woods
CG. 1999. The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13.1-13.2.
Eur.J.Hum.Genet. 7:815 820.

Roberts E, Hampshire DJ, Springell K, Pattison L, Y Crow, Jafri H, Corry P, Kabani G, Mannon J, Rashid Y, Keen J, Bond J,
Woods CG. 2002. Autosomal recessive primary microcephaly: an analysis of locus heterogeneity and phenotypic variation.
J.Med.Genet. 39:718 721.

Roche AF, Mukherjee D, Guo S, Moore W. 1987. Head Circumference Data: Birth to 18 Years. Pediatrics 79:706712.

Trimborn M, Bell SM, Felix C, Rashid Y, Jafri H, Griffiths PD, Neumann LM, Krebs A, Reis A, Sperling K, Neitzel H,
Jackson AP. 2004. Mutations in microcephalin cause aberrant regulation of chromosome condensation. Am.J.Hum.Genet.
75:261266.

Woods CG, Bond J, Enard W. 2005. Autosomal Recessive Primary Microcephaly (MCPH): A Review of Clinical, Molecular,
and Evolutionary Findings. Am.J.Hum.Genet. 76:717728.