A review on microcephaly genes

Introduction. Microcephaly is a neurological disorder [1] defined as a small cranium with a significantly reduced head circumference (HC) of more than two standard deviations (SD) [2, 3]. Microcephaly can occur with congenital (primary) or postnatal (secondary) onset, and is caused by genetic or non-genetic factors [4, 5]. Autosomal recessive primary microcephaly is characterized by non-progressive mild-to-moderate mental retardation and a substantial reduction in prenatal human brain growth without alteration of the cerebral architecture [6–9]. It is a model disease to study human neurogenesis [10]. Secondary, microcephaly is a disorder in which the brain is of the expected size at birth but subsequently fails to grow normally. The condition occurs due to mutations in an apparently ubiquitously expressed X-linked gene MeCP2 which has a crucial role in maintaining the mature neuronal state [11]. The incidence is 1 in 10000 in consanguineous populations, less in non-consanguineous populations [7]. Microcephaly genes. Clinical surveys from the 1950s and earlier suggested that microcephaly was likely to be an autosomal recessive Mundelein disorder. However, molecular studies of this disorder were performed in the late 1990’s [12]. Microcephaly exhibits genetic heterogeneity, having been associated with mutations in seven microcephaly loci. These loci, known as «microcephaly genes», are, therefore, presumed to play important roles in size regulation during brain development [13]. So far, seven genetic loci (MCPH1-7) for microcephaly have been mapped [14]. Genetic causes of microcephaly subtypes 1–7 include mutations in genes encoding microcephalin (MCPH1) [15], WDR62 (MCPH2) [8], CDK5RAP2 (MCPH3) [16], CEP152 (MCPH4) [39], ASPM (MCPH5) [17], CENPJ (MCPH6) [16], STIL (MCPH7) [18]. A detailed description of the MCPH gene mutations and their most likely functions in the development of microcephaly is as follows (Table 1). Microcephalin (MCPH1). Microcephalin (MCPH1) is an 835 amino acid protein containing BRCA1 C-terminal (BRCT) domains. MCPH1 gene in chromosome locus 8p23 contains 14 exons, the genomic size is 241905 bp with three isoforms reported so far and 8032 bp Open Reading Frame (ORF) [15]. The mutations identified earlier were truncating mutations and, therefore, were predicted to lead to a complete loss of the gene function [19]. Jackson and colleagues has refined the MCPH1 region from 13cM to 2.1Mb. In two consanguineous families of northern Pakistani origin, a single homozygous 74CG (S25X) non-sense mutation was identified in the second exon of a 2.505 nucleotide, 14 exon of uncharacterized gene [15] (Figure, a) which encodes the protein microcephalin, also is referred to as ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 3. P. 181–189


Introduction.
Microcephaly is a neurological disorder [1] defined as a small cranium with a significantly reduced head circumference (HC) of more than two standard deviations (SD) [2,3].Microcephaly can occur with congenital (primary) or postnatal (secondary) onset, and is caused by genetic or non-genetic factors [4,5].Autosomal recessive primary microcephaly is characterized by non-progressive mild-to-moderate mental retardation and a substantial reduction in prenatal human brain growth without alteration of the cerebral architecture [6][7][8][9].It is a model disease to study human neurogenesis [10].Secondary, microcephaly is a disorder in which the brain is of the expected size at birth but subsequently fails to grow normally.The condition occurs due to mutations in an apparently ubiquitously expressed X-linked gene MeCP2 which has a crucial role in maintaining the mature neuronal state [11].The incidence is 1 in 10000 in consanguineous populations, less in non-consanguineous populations [7].
Microcephalin (MCPH1).Microcephalin (MCPH1) is an 835 amino acid protein containing BRCA1 C-terminal (BRCT) domains.MCPH1 gene in chromosome locus 8p23 contains 14 exons, the genomic size is 241905 bp with three isoforms reported so far and 8032 bp Open Reading Frame (ORF) [15].The mutations identified earlier were truncating mutations and, therefore, were predicted to lead to a complete loss of the gene function [19].Jackson and colleagues has refined the MCPH1 region from 13cM to 2.1Mb.In two consanguineous families of northern Pakistani origin, a single homozygous 74CG (S25X) non-sense mutation was identified in the second exon of a 2.505 nucleotide, 14 exon of uncharacterized gene [15] (Figure , a) which encodes the protein microcephalin, also is referred to as BRIT1 for BRCT Repeat Inhibitor of hTert expression [20].Later, the mutation in this gene was reported at disorder of microcephaly, short stature and misregulated chromosome condensation, known as Premature Chromosome Condensation Syndrome (PCC).It was therefore discovered that both MCPH1 and PCC syndrome are allelic disorders originated from mutations within the same gene [21].A missense mutation has been reported in MCPH1 gene demonstrating less severe cellular phenotype and mild microcephaly [22].Also, a large deletion mutation encompassing first 6 exons of Microcephalin gene in an Iranian family, showing autosomal recessive mental retardation and mild microcephaly, has been reported [23].Loss of function mutations in the genes associated with MCPH reduced human brain size by about two-thirds, without producing gross abnormalities in brain organization or physiology [24].RNA interference (RNAi) of MCPH1 has implicated the corresponding protein as a DNAdamage response protein that regulates the transcription of Chk1 and BRCA1, two genes involved in the response to DNA damage.MCPH1 has a function downstream of Chk1 in the ATR-signalling pathway.MCPH1 also plays an ATR-independent role in maintaining inhibitory Chk1 phosphorylation, which prevents premature entry into mitosis [25].Farooq et al. described the Craniosynostosis-Microcephaly with Chromosomal Breakage (CMCB) and other abnormalities that were caused by a truncating mutation (302CG) in exon 4 of the MCPH1 gene.CMCB is found to be allelic to PCC syndrome and MCPH1 [26].Most reported mutations induce the production of non-functional, truncated proteins [2].Microcephalin has a role in DNA damage response [27,28] and regulation of cell cycle checkpoints [29].It has been proposed that mutations in microcephalin are a causal link between impaired DNA damage response signaling and microcephaly and that misregulated chromosome condensation in MCPH1 is mediated by condensin II protein [30,31].It has been demonstrated that microcephalin protein is recruited to double-strand breaks via interaction of its C-terminal BRCT domains with phosphorylated H2AX, a histone variant that marks sites of damaged DNA, further expanding the signal to Chk1 and Chk2 via subsequent phosphorylation, leading to the modulation of cell cycle, DNA repair and apoptosis [31].WDR62 (MCPH2).WDR62 gene was identified as the second most common cause of MCPH after finding homozygous missense and frame-shifting mutations in seven MCPH families [8].The gene lies within the MCPH2 candidate linkage interval at chromosome 19q13.12with 32 functional exons and genomic size of 50230 bp [21].The gene has two predicted isoforms and the protein consisting of 1523 residues contains 15 WD repeats.In three recently published research articles, authors have found six different homozygous recessive mutations in human WDR62 showing linkage at MCPH2 locus [8,33,34].Interestingly, four of them were missense mutations.Like other MCPH proteins overexpressed WDR62 was localized to the spindle poles during mitosis.In contrast, a mutant WDR62 protein, carrying one of the causative missense alterations, p.Arg438His, failed to localize in the spindle poles during mitosis [8].Bilguvar et al. have found 5 homozygous WDR62 mutations including deletions and premature terminations in patients suffering from microcephaly and some additional phenotypes, belonging to Turkish origin [33].WDR62 missense mutations might impair the centrosomal function of WDR62, whereas, on the other hand, a complete loss of WDR62 function might affect severely neuronal migration as well.Taken together, the current studies strongly suggested a function for WDR62 in proliferation and spindle formation, as well as in neuronal migration, thereby showing a com-mon mechanism underlying primary microcephaly and malformations of cortical development [5].Yu et al. also identified six families with syndrome of congenital microcephaly and diverse defects in cerebral cortical architecture.They identified six mutations in WDR62 gene by use of targeted high throughput sequence analysis [34].Nuclear localization of WDR62 in human neurons that had just migrated to the cortical plate was also observed which suggested that WDR62 might play an important role in cortical lamination as well [8].
CDK5RAP2 (MCPH3).The cyclin dependent kinase 5 regulatory subunit-associated protein 2 is a protein that in humans is encoded by the CDK5RAP2 gene, also referred to as the centrosome associated protein 215 (CEP215) or the CDK5 activator binding protein C48 (C48).
Four isoforms have been reported so far.The human full length, 1893 amino acid CDK5RAP2 protein contains an N-terminal interaction site with the gamma tubulin ring complex (gTuRC), a C-terminal interaction site with CDK5 regulatory subunit1 lines and tumors, and its increased expression is positively associated with proliferation of glio blastoma cells [2,35].The MCPH3 locus was refined from 12 cM to 2.2 Mb by the identification from a family of northern Pakistani origin [16].The gene contains 38 exons; genomic size is 191290 bp, with 5682 bp ORF [2,16,36].Multiple transcript variants exist for this gene on chromosome 9q33.2,but the complete molecular mechanism for only two has been determined [14].A homozygous 243T ®A (S81X) nonsense mutation and an intronic mutation 15 bases prior to the normal splice acceptor sequence Mutations reported in (a) MCPH1 and (b) ASPM.A micro deletion that includes the promoter region and first nine exons of MCPH1 is indicated by a horizontal line.The translocation break point in the ASPM gene is depicted by a vertical line.Recurrent mutations are denoted by an additional star [12] IVS26-15AG (E385fsX4) were identified.Both mutations are segregated from the disease within the families and were not present in 1380 control alleles.The intronic mutation created a new, superior splice acceptor site in exon 27, leading to the addition of four new amino acids and a termination codon [16].
Hassan et al. described a previously known mutation (S81X) in MCPH family from Northern Pakistan and concluded that the exact nomenclature for this mutation was 246T ® A (Y82X) [37].Zhang et al. demonstrated that CDK5RAP2 is required for spindle checkpoint regulation.They found that loss of its function leads to chromosome mis-segregation and reduced expression of spindle checkpoint proteins, via interaction with their promoters and transcription regulation [38].
CEP152 (MCPH4).Guernsey et al. have discovered both homozygous and heterozygous mutations in CEP152 gene located within MCPH4 linkage locus at chromosome 15q21.1 in two Canadian families with MCPH using single nucleotide polymorphism (SNP)based genome scan.However, in mice, they were able to generate a cDNA clone product of the correct size spanning exons 21-24 of the mouse CEP152 ortholog by using RNA extracted from mouse brain tissue; cDNA could be readily detected in stage 12.5 and 14.5 embryonic brains.They have also used tools of bioinformatics to elaborate the pathogenic effect of homozygous mutation in CEP152 gene (Q265P) in two MCPH4 linked families [39].Blachon et al. have demonstrated that Asl (ortholog of CEP152 in Drosophila) is a conserved gene required for ciliogenesis; which is involved in the initiation of centriole duplication and that it is dispensable for pericentriolar material (PCM) recruitment in meiosis in MC centrioles (Mother Centrioles).They were successful to show that Asl is essential for centriole formation in brain cells [40].A homozygous MCPH-causing mutation was reported in human CEP63.CEP63 forms a complex with another MCPH protein, CEP152, a conserved centrosome duplication factor.Together, these two proteins are essential for maintaining normal centrosome numbers in cells.Using super-resolution microscopy, it was found that CEP63 and CEP152 co-localize in a discrete ring around the proximal end of the parental centriole, a pattern specifically disrupted in CEP63-deficient cells derived from patients with MCPH.This work suggests that the CEP152-CEP63 ring-like structure ensures normal neurodevelopment and that its impairment particularly affects human cerebral cortex growth [9].
ASPM (MCPH5).The most common cause of MCPH is homozygous mutations in the MCPH5 gene [41] (Figure , b), Abnormal Spindle-like, Microcephaly associated gene (ASPM) [17,[42][43][44][45]. ASPM is a very large protein of 3477 amino acids comprising a putative microtubule-binding domain at its N-terminal [46,47], a calponin homology domain, multiple IQ calmodulin binding domains [46,48] and a carboxy terminal region with no identifiable domains [19, 48,49].ASPM is a large gene with 28 exons and a 10.4-kb ORF.The 24 published ASPM mutations comprise nonsense mutations, deletions of 1-7 base pairs, a break point translocation, and intronic splice-donor point mutations [42][43][44][45].The mutations are distributed throughout the gene and are all predicted to produce truncated protein products ranging in size from 116 amino acids to 3,357 amino acids [42,43].It has been suggested that the common mechanism of action of ASPM mutation is nonsense-mediated mRNA decay [50] Nonsense-mediated mRNA decay is a mechanism that selectively degrades mRNAs harboring premature termination (nonsense) codons [72].ASPM hampers symmetric cell division of neural progenitors [51].It has been suggested that a second gene causing MCPH exists at the MCPH5 locus, as mapping studies in five Jordanian and Dutch families linked the families to the MCPH5 locus but excluded the genetic region containing ASPM [52].Sadi et al. identified heterozygous ASPM mutations in three Algerian siblings born to consanguineous parents of which two were nonsense and frameshift mutations predicting a loss of function either by nonsense-mediated mRNA decay or by synthesis of a severely truncated and presumably inactive ASPM protein.ASPMrelated MCPH is associated with simplified cortical gyration [32].The function of the ASPM protein in neural progenitor cell expansion, as well as its localization to the mitotic spindle and mid body, suggested that it regulates brain development by a cell division-related mechanism [49].In some recent studies, expression of ASPM in cancer cells has been investigated.ASPM has been found down-regulated in ionizing-radiation treated cells [53] and its down-regulation has shown to decrea-se the Brca1 protein expression as well [54].ASPM plays a role in mitotic spindle function including orientation of cleavage plane.The spindle apparatus dictates the plane of cell cleavage, which is critical in the choice between symmetric or asymmetric division [55].
CENPJ (MCPH6).The human centromeric protein J encoded by gene CENPJ, also referred to as centrosomal P4.1-associated protein (CPAP), LAG-3-associated protein (LAP) or LYST-interacting protein1 (LIP1).The human full length CENPJ protein of 1338 amino acid contains a 112-amino acid long microtubule destabilizing motif PN2-3 [56] and two C-terminal 14-3-3 binding sites [57].The CENPJ gene (MIM 609279) spans 40672 bp with 5187 bp ORF.Four mutations have been reported in this gene so far.Initially two mutations were reported in three families [16] including two families from Northern Pakistan having one mutation and other from Brazil with second mutation within a previously identified locus on human chromosome 13q12.2[58].The 6-Mb MCPH6 region was refined by the use of novel polymorphic microsatellite markers that were genotyped in the original Brazilian family used to map the locus and in two additional northern Pakistani affected consanguineous families [16].This facilitated the reduction of the locus to 3.1 Mb and enabled the selection of CENPJ gene in which two homozygous mutations were identified: the frameshift mutation 17 del Cin exon 2 (T6fsX3), in two pedigrees, and the missense mutation 3704A ® T in exon 16 (E1235V), in one pedigree.3704A ® T is the only MCPH mutation described that is not predicted to result in a protein truncation.The E1235V mutation occurs in a highly conserved Tcp10 binding domain, which is involved in binding to the protein 4.1R-135 [4].Later, in a Pakistani family, a deletion of four consecutive nucleotides (TCAG) was identified in exon 11 of CENPJ gene, leading to frameshift and premature termination by a codon 19 bp downstream in the same exon, predicted to add six amino acids downstream of the mutation [59].More recently, a novel splicing mutation in CENPJ gene [60] has been observed in Seckel syndrome patients.CENPJ is localized at the centrosome during mitosis and it is concentrated at the mitotic spindle poles during pro-metaphase and metaphase [16,56].Cho et al. have observed that depletion of CENPJ disrupts centrosome integrity, and cells lacking CPAP are arrested in mito-sis with multipolar spindles.It has been found that CENPJ depletion by the use of siRNA lead to an increase of multiple spindle poles, mitosis arrest and apoptosis [61].CENPJ interacts with the N-terminal domain of the p65 subunit (RelA) of NF-kappa B, a transcription factor important for various cellular events such as inflammation, immune response, proliferation and apoptosis [62].
SIL/STIL (MCPH7).The human SCL/TAL1-interrupting locus gene encodes STIL, also referred to as SCL-interrupting locus (SIL).STIL is an immediate early gene that encodes a cytosolic protein of 150 kDa.It contains 20 exons and 1287 residues in full-length protein [2,18,63].Two isoforms have been reported.The STIL gene spans 63018 bp with 5225 bp ORF.Sequence analysis of STIL revealed 3 different homozygous protein truncating mutations in affected members of 5 Indian families linked to MCPH7.Heterogeneity analysis of 24 families previously excluded from linkage to the six known MCPH loci suggested linkage of five families (20.83 %) to the MCPH7 locus.Further, another ascertained family was homozygous for the same mutation as one of the original families [18].It is also reported that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH [64].Karkera et al. found that the human STIL protein contains a putative nuclear localization signal and a cysteine-terminal domain similar to the C-terminal domain of TGF-beta [63].STIL is an early response gene that is ubiquitously expressed in proliferating cells and during early embryonic development [65,66].STIL is expressed throughout the cytosol with increased expression in the perinuclear region that likely plays a role in mitotic entry (cell cycle progression G2-M), apoptosis control and centrosome function [18].
Mutation Continuum of Microcephaly Genes in Pakistan population.Due to high rate of consanguinity (~61 %) and large family size, Pakistani population has proved to be rich for genetic linkage studies.A total of about 95 families have been reported with mutations in any of microcephaly genes from Pakistan (Table 2).Except one recently discovered locus from India (MCPH7), all other MCPH loci have been reported in Pakistani families.ASPM gene mutations have been found in 83 families with 2 families having compound heterozygous mutations.Two mutations 3978G ® A and 8508delGA were most common, segregating in 18 and 4 families, respectively.CDK5RAP2 gene mutations have been found in three Pakistani families so far, and they are exclusive to Pakistan.One mutation (IVS26-15A ® G) was observed in one family while other mutation (Y82X) was present in two families.Two mutations (17delC and 3243delTCAGa) have been found in CENPJ gene segregating in three Pakistani families [14].In one Pakistani family, linked to MCPH2 locus, a missense mutation 3232G ® A (Ala1078Thr) in exon 27 of the WDR62 gene has been detected [8].Previously the locus was reported in Pakistani families only but the mutations in WDR62 gene have been found in microcephaly patients from other countries as well [8,33,34].
Mutations in genes encoding not only mitotic and apoptotic proteins but also transcription factors may be responsible for malformative microcephaly syndromes [68].CASK is a multidomain scaffolding protein that interacts with the transcription factor TBR1 and regulates expression of genes involved in cortical development.It has been reported that all affected individuals with CASK mutations had congenital or postnatal microcephaly [69].The common variations in the microcephaly genes do not affect the risk of Alzheimer Disease or that their effect size is small [70].It was suggested that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer [71].
Conclusion.The proteins encoded by the microcephaly genes are involved in various molecular mechanisms including cell division, cell cycle regulation, appropriate spindle orientation, DNA damage response signaling, microtubule dynamics, etc.Recent functional studies have confirmed the presence of microcephaly proteins at centrosome for at least part of the cell cycle.Therefore, it may be concluded that centrosome is the final integration point for many regulatory pathways affecting prenatal neurogenesis in mammals.Better genotyping, neuro-imaging and neuro-physiological testing, along with constitution of genetically homogeneous groups of patients, would help in establishing exact genotype-phenotype correlation.Mutation screening in microcephaly families is need of the time.It will help in genetic counselling and prenatal diagnosis for microcephaly, and thus in turn may help reducing the incidence of mirocephaly from highly consanguineous population.