Primary microcephaly is thought to result from genetic defects of the developmental program that generates large brain hemispheres in humans. Autosomal recessive inheritance is likely in most familial cases, and four loci were recently mapped by homozygosity. Homozygosity mapping of a new locus, MCPH5, is reported in this study, with a maximum multipoint LOD score of 3.51 at marker D1S1723, in a family of Turkish origin. The minimal critical region spans 11.4 cM between markers D1S384 and D1S2655, at 1q25-q32, and encompasses the cytogenetic breakpoints of chromosomal aberrations previously reported in unrelated patients with microcephaly (Jamieson, 2000).
Primary microcephaly is a genetic disorder in which an affected individual has a head circumference >3 SDs below the age- and sex-related mean. A small but apparently normally formed brain is the reason for the reduced head circumference, and, probably because of this, all affected individuals are mentally retarded. The condition is genetically heterogeneous, and four loci have already been identified. A fifth locus, MCPH5, is reported that is an 8-cM region mapping to chromosome 1q31, defined by the markers GATA135F02 and D1S1678 (Pattison, 2000).
Locus heterogeneity is well established in autosomal recessive primary microcephaly (MCPH) and to date five loci have been mapped. However, the relative contributions of these loci have not been assessed and genotype-phenotype correlations have not been investigated. A study population of 56 consanguineous families resident in or originating from northern Pakistan was ascertained and assessed. A panel of microsatellite markers spanning each of the MCPH loci was designed, against which the families were genotyped. The head circumference of the 131 affected subjects ranged from 4 to 14 SD below the mean, but there was little intrafamilial variation among affecteds. MCPH5 was the most prevalent, with 24/56 families consistent with linkage; 2/56 families were compatible with linkage to MCPH1, 10/56 to MCPH2, 2/56 to MCPH3, none to MCPH4, and 18/56 did not segregate with any of the loci. It is concluded that MCPH5 is the most common locus in this population. On clinical grounds alone, the phenotype of families linked to each MCPH locus could not be distinguished. Further MCPH loci await discovery with a number of families as yet unlinked (Roberts, 2002).
One of the most notable trends in mammalian evolution is the massive increase in size of the cerebral cortex, especially in primates. Humans with autosomal recessive primary microcephaly (MCPH) show a small but otherwise grossly normal cerebral cortex associated with mild to moderate mental retardation. Genes linked to this condition offer potential insights into the development and evolution of the cerebral cortex. The most common cause of MCPH is homozygous mutation of ASPM, the human ortholog of the Drosophila abnormal spindle gene, which is essential for normal mitotic spindle function in embryonic neuroblasts. The mouse gene Aspm is expressed specifically in the primary sites of prenatal cerebral cortical neurogenesis. Notably, the predicted ASPM proteins encode systematically larger numbers of repeated 'IQ' domains between flies, mice and humans, with the predominant difference between Aspm and ASPM being a single large insertion coding for IQ domains. These results and evolutionary considerations suggest that brain size is controlled in part through modulation of mitotic spindle activity in neuronal progenitor cells (Bond, 2002).
MCPH exhibits genetic heterogeneity associated with mutations in five recessive loci that cause clinically indistinguishable disorders. Of these, mutations in MCPH5 (1q31) are the most prevalent, present in 24 of the 56 consanguineous Northern Pakistani families affected with MCPH. MCPH5 is also the only locus associated with MCPH known to be affected in multiple ethnic groups. Brains of individuals with MCPH5 mutations show a substantial reduction in size of the cerebral cortex without evidence of abnormal neuronal migration or abnormal architecture (Bond, 2002).
A positional cloning strategy was adopted to identify the gene MCPH5, using the 24 consanguineous Northern Pakistani families (including 61 affected individuals aged 4-35 years) affected with MCPH linked to the MCPH5 locus. A complete electronic contig was assembled of a 8-Mb region of 1q31 containing MCPH5 by overlapping BAC clones and transcripts where sequences were available in the public domain. New polymorphic microsatellite markers were designed and used to locate crossovers in three families with more than one affected individual, including the family in which the locus was previously defined. Each of these families had a two-point lod score >3 for probability of linkage to the MCPH5 locus. This reduced the gene region for MCPH5 from 8 Mb to 2.2 Mb. More polymorphic microsatellite markers at a density of roughly one per 100 kb were then used to determine the genotypes of all 24 families. A region of 600 kb was identified in which one haplotype was shared between three different families and a second haplotype was shared by three other families, indicating common ancestors carrying mutations in MCPH5. This region contained four potential genes identified from genetic databases. One, CRB1, was eliminated because mutations in that gene cause blindness due to Leber congenital amaurosis without microcephaly. The other three transcripts, which are expressed in fetal brain, included a homolog of the KIAA1089 gene on chromosome 9q34, a zinc-finger gene and a number of transcripts with similarity to asp. The complete sequences of these three genes were determined and they were sequenced in the four largest families mapping to MCPH5, each of which had a distinct haplotype (Bond, 2002).
A homozygous mutation was identified introducing a premature stop codon into the predicted ASPM (abnormal spindle-like microcephaly associated) open reading frame in each of the four affected families. The four mutations are 719-720delCT (exon 3), causing a frameshift leading to a premature termination 15 codons downstream; 1258-1264 delTCTCAAG (exon 3), causing a frameshift leading to a premature termination 31 codons downstream; 7761T-->G (exon 18), producing immediate truncation, and 9159delA (exon 21), causing a frameshift leading to a premature termination 4 codons downstream. Each mutation segregated with the disease as expected within the families, and was not found in 200 normal control chromosomes. It was not possible to distinguish between the phenotypes of the four families in which mutations were found (Bond, 2002).
The predicted 10,434-bp open reading frame of ASPM was confirmed in overlapping segments of cDNA from fetal brain and colon. The Kozak start and polyadenylation sites are consensus sequences. Exon 4 and large segments of exons 3 and 18 were not found in previously reported human expressed sequence tags (ESTs) or mRNA. The gene contains 28 exons and spans 62 kb of genomic sequence on the BAC RP11-32D17 (Bond, 2002).
Interspecies comparisons of the predicted abnormal spindle proteins indicate that they are notably conserved overall, but show a consistent correlation of greater protein size with larger brain size. Both the putative amino-terminal microtubule-binding region of Asp and a putative calponin-homology domain (Saunders, 1997) are conserved in Aspm and ASPM. Most of the remainder of the protein encodes a series of repeated calmodulin-binding IQ domains, and the number of these repeats varies markedly between species. The closest potential Caenorhabditis elegans homolog encodes 2 IQ repeats, whereas Asp encodes 24 repeats; Aspm contains 61 repeats and ASPM 74 repeats, the largest number of IQ domains reported in any protein. The size difference between Aspm and ASPM principally reflects an insertion of roughly 900 bp into exon 18, resulting in a large exon of 4.7 kb and adding ten IQ domains to the predicted protein. BLAST analysis of this inserted sequence shows that its closest match is to an upstream region of exon 18, suggesting that the human insertion may have arisen from a duplication. There are other, smaller potential insertions that distinguish ASPM from Aspm, predominantly involving the two largest exons, 3 and 18. Although the function of the repeated IQ domains is not known, loss of the terminal 425 amino acids of ASPM (mutation 9159delA), which encompass six IQ domains and the carboxy-terminal region, is sufficient to cause MCPH, suggesting that this region is essential for normal brain growth (Bond, 2002).
Northern-blot analysis of Aspm shows that it is highly expressed at embryonic day (E) 11-17 and encoded on a large transcript (>9.5 kb). In situ hybridization of an antisense probe to Aspm shows preferential expression during cerebral cortical neurogenesis, specifically in the cerebral cortical ventricular zone at E14.5 and E16.5. Expression is also observed in the proliferative region of the medial and lateral ganglionic eminence (MGE and LGE) at both E14.5 and E16.5, and in the ventricular zone of the dorsal diencephalon at E14.5, with weak hybridization in this region at E16.5. Other regions of the dorsal diencephalon show Aspm expression at E14.5, but by E16.5, expression is increasingly limited to telencephalic structures. Little or no hybridization was observed in ventral diencephalon and more caudal parts of the brain. Aspm expression is quite intense at E14.5, when there are many progenitor cells in the cortical ventricular zone, but expression has already begun to decrease in intensity by E16.5. Fetal Aspm expression is overall greatest in the ventricular zones, which contain the progenitor cells for cerebral cortical pyramidal neurons. Aspm is also highly expressed in the MGE and LGE, where up to one third of cerebral cortical neurons, predominantly interneurons, are formed and subsequently migrate non-radially from the ventral telencephalon into the cerebral cortex. Expression of Aspm is greatly reduced by postnatal day (P) 0 (day of birth), when neurogenesis in the cortical ventricular zone is completed and gliogenesis is increasing, suggesting that Aspm is preferentially expressed in progenitors that produce neurons rather than glia. By P9, expression is limited to rare scattered cells in the neocortex, though expression continues in regions of the brain that show persistent postnatal neurogenesis, such as the dentate gyrus and the subventricular zone of the rostral migratory stream leading to the olfactory bulb. These data suggest that Aspm has a preferential role in regulating neurogenesis before and after birth, and that the pattern of brain malformation associated with mutations in MCPH5 (primarily reduction in size of the cerebral cortex) closely parallels the overall distribution of Aspm expression (Bond, 2002).
The size of human brain tripled over a period of ~2 million years (MY) that ended 0.2-0.4 MY ago. This evolutionary expansion is believed to be important to the emergence of human language and other high-order cognitive functions, yet its genetic basis remains unknown. An evolutionary analysis of genes controlling brain development may shed light on it. ASPM (abnormal spindle-like microcephaly associated) is one such gene; nonsense mutations lead to primary microcephaly, a human disease characterized by a 70% reduction in brain size. Evidence is provided suggesting that human ASPM went through an episode of accelerated sequence evolution by positive Darwinian selection after the split of humans and chimpanzees but before the separation of modern non-Africans from Africans. Because positive selection acts on a gene only when the gene function is altered and the organismal fitness is increased, these results suggest that adaptive functional modifications occurred in human ASPM and that it may be a major genetic component underlying the evolution of the human brain (Zhang, 2003).
Evidence is provided that advantageous amino acid substitutions unrelated to IQ repeats have been fixed by adaptive selection in human ASPM after the human-chimpanzee split, which strongly suggests that ASPM might be an important genetic component in the evolutionary expansion of human brain. The episode of positive selection on ASPM appears to have ended some time ago, since there is no evidence for positive selection on ASPM in current human populations; rather, relatively strong purifying selection is detected. Roughly, selective sweeps occurring in the past 0.5N generations may be detected, where N is the effective population size of humans and is thought to be ~10,000. That is, the positive selection detected in ASPM occurred some time between 6-7 and 0.1 MY ago (0.5 x 10,000 generations x 20 years/generation). The latter date coincides with the suggested time of migration of modern humans out of Africa. It is also interesting to note that although the precise time when positive selection acted on ASPM is difficult to pinpoint, the given estimate is consistent with the current understanding that the human brain expansion took place between 2-2.5 and 0.2-0.4 MY ago. Furthermore, a selective sweep in human FOXP2, a gene involved in speech and language development, has been detected. This sweep was estimated to have occurred no earlier than 0.1-0.2 MY ago. That is, the adaptive evolution of FOXP2 postdated that of ASPM, consistent with the common belief that a big brain may be a prerequisite for language (Zhang, 2003).
Studies of ASPM in model organisms can help in the understanding of how it impacts brain size. The mouse Aspm is highly expressed in the embryonic brain, particularly during cerebral cortical neurogenesis. The fruit fly ortholog asp is involved in organizing and binding together microtubules at the spindle poles and in forming the central mitotic spindle. Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development. The amino acid substitutions in human ASPM are located in exons 3, 18, 20, 21, and 22, which encode a putative microtubule-binding domain and an IQ calmodulin-binding domain. These features suggest that the adaptive substitutions in human ASPM might be related to the regulation of mitosis in the nervous system, which can be tested in the future by functional assays of human ASPM as well as a laboratory-reconstructed ASPM protein of the common ancestor of humans and chimpanzees (Zhang, 2003).
A prominent trend in the evolution of humans is the progressive enlargement of the cerebral cortex. The ASPM (Abnormal spindle-like microcephaly associated) gene has the potential to play a role in this evolutionary process, because mutations in this gene cause severe reductions in the cerebral cortical size of affected humans. The evolution of ASPM is significantly accelerated in great apes, especially along the ape lineages leading to humans. Additionally, the lineage from the last human/chimpanzee ancestor to humans shows an excess of nonsynonymous over synonymous substitutions, which is a signature of positive Darwinian selection. A comparison of polymorphism and divergence using the McDonald-Kreitman test confirms that ASPM has indeed experienced intense positive selection during recent human evolution. This test also reveals that, on average, ASPM fixed one advantageous amino acid change in every 300,000-400,000 years since the human lineage diverged from chimpanzees. It is therefore concluded that ASPM underwent strong adaptive evolution in the descent of Homo sapiens, which is consistent with its putative role in the evolutionary enlargement of the human brain (Evans, 2004).
How might the molecular evolution of ASPM contribute to the progressive enlargement of the cerebral cortex? There is currently no direct data on the biochemical function of ASPM in mammals. However, the Drosophila homolog, asp, has been studied extensively, and has been shown to have a critical function in organizing the mitotic and meiotic spindle structures. During the development of the brain, the proliferating progenitor cells of the neuroepithelium can undergo two modes of cell divisions: symmetric divisions with mitotic spindles in the plane of the neuroepithelium to generate two identical progenitor cells, and asymmetric divisions with mitotic spindles perpendicular to the plane of the neuroepithelium to generate one progenitor and one neuron. The regulation of mitotic spindle orientations (and hence the proportions of symmetric versus asymmetric cell divisions) in the proliferating neuroepithelium is thought to have a profound impact on the final size of the cerebral cortex, with higher proportions of symmetric divisions during early neuroepithelium development corresponding to larger cortex. Based on mouse data, ASPM is expressed specifically in the developing neuroepithelium. If the mammalian ASPM protein, like its Drosophila homology, is also involved in the organization of mitotic spindle structures, then it is conceivable that alterations to the amino acid sequence (and hence the biochemical properties) of ASPM during evolution could affect the regulation of mitotic spindle orientation in proliferating cells of the neuroepithelium. This in turn could alter the size of the cerebral cortex. These possibilities are potentially testable in future studies (Evans, 2004).
Autosomal recessive primary microcephaly (MCPH) is a neural developmental disorder in which patients display significantly reduced brain size. Mutations in Abnormal Spindle Microcephaly (ASPM) are the most common cause of MCPH. This study investigated the underlying functions of Aspm in brain development; Aspm expression was found to be critical for proper neurogenesis and neuronal migration. The Wnt signaling pathway is known for its roles in embryogenesis, and genome-wide siRNA screens indicate that ASPM is a positive regulator of Wnt signaling. Knockdown of Aspm results in decreased Wnt-mediated transcription, and expression of stabilized β-catenin can rescue this deficit. Coexpression of stabilized β-catenin can rescue defects observed upon in vivo knockdown of Aspm. These findings provide an impetus to further explore Aspm's role in facilitating Wnt-mediated neurogenesis programs, which may contribute to psychiatric illness etiology when perturbed (Buchman, 2011).
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