Copyright 2003 by the Genetics Society of America Evolution of the Human ASPM Gene, a Major Determinant of Brain Size Jianzhi Zhang1 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109 Manuscript received July 7, 2003 Accepted for publication August 20, 2003 ABSTRACT .million years (MY) that ended 0.2–0.4 MY ago 2ف The size of human brain tripled over a period of 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 of such genes, as nonsense mutations lead to primary microcephaly, a human disease characterized by a 70% reduction in brain size. Here I provide evidence 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, my 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. MONG mammals, humans have an exceptionally big The reduction in head circumference correlates with a A brain relative to their body size. For example, in markedly reduced brain size. Microcephaly is genetically comparison with chimpanzees, the brain weight of hu- heterogeneous, associated with mutations in at least five mans is 250% greater while the body is only 20% heavier loci (Mochida and Walsh 2001; Kumar et al. 2002), (McHenry 1994). The dramatic evolutionary expan- one of which was recently identified and named ASPM sion of the human brain started from an average brain (abnormal spindle-like microcephaly associated; Bond million years (MY) ago and et al. 2002). Four different homozygous mutations in 2.5–2ف weight of 400–450 g MY ago ASPM introducing premature stop codons were found 0.4–0.2ف g 1450–1350ف ended with a weight of (McHenry 1994; Wood and Collard 1999). This pro- to cosegregate with the disease in four respective fami- cess represents one of the most rapid morphological lies, while none of these mutations were found in 200 changes in evolution. It is generally believed that the normal human chromosomes (Bond et al. 2002). Be- brain expansion set the stage for the emergence of cause the brain size of a typical microcephaly patient human language and other high-order cognitive func- (430 g; Mochida and Walsh 2001; Kumar et al. 2002) tions and that it was caused by adaptive selection (Decan is comparable with those of early hominids such as the 1992), yet the genetic basis of the expansion remains 2.3- to 3.0-MY-old Australopithecus africanus (420 g; elusive. A study of human mutations that result in unusu- McHenry 1994; Wood and Collard 1999), I hypothe- ally small brains may help identify the genetic modifica- size that ASPM may be one of the genetic components tions that contributed to the human brain expansion. underlying the human brain expansion. Signatures of In this regard, primary microcephaly (small head) is of accelerated evolution of ASPM under positive selection particular interest (Mochida and Walsh 2001; Bond during human origins would strongly support my hy- et al. 2002; Kumar et al. 2002). Microcephaly is an autoso- pothesis, because the action of positive selection indi- mal recessive genetic disease with an incidence of 4–40 cates a modification in gene function resulting in ele- per million live births in western countries (Mochida vated organismal fitness (Zhang et al. 2002). Below I and Walsh 2001; Kumar et al. 2002). It is defined as a provide population genetic and molecular evolutionary head circumference Ͼ3 standard deviations below the evidence for the operation of such adaptive selection population age-related mean, but with no associated on ASPM. malfunctions other than mild-to-moderate mental retar- dation (Mochida and Walsh 2001; Kumar et al. 2002). MATERIALS AND METHODS Sequencing of ASPM: The human ASPM gene has 28 exons. Sequence data from this article have been deposited with the All 28 exons were PCR amplified from genomic DNA samples EMBL/GenBank Data Libraries under accession nos. AY367065–87. of 14 human (Homo sapiens) individuals of different geo- 1Address for correspondence: Department of Ecology and Evolutionary graphic origins (2 African Pygmies, 3 African Americans, 4 Biology, University of Michigan, 3003 Natural Science Bldg., 830 N. Europeans, 2 Southeast Asians, 1 Chinese, 1 Pacific islander, University Ave., Ann Arbor, MI 48109. E-mail: [email protected] and 1 South American), using the high-fidelity Taq of In- Genetics 165: 2063–2070 (December 2003) 2064 J. Zhang vitrogen (Carlsbad, CA). The PCR products were then purified site/year, which was also estimated from a genomic compari- and sequenced in both directions. Polymorphisms that appear son between the human and chimpanzee (Britten 2002). I only once (singletons) were confirmed by a second PCR- assumed that all indels with sizes that are multiples of three sequencing experiment. The human DNA samples were pur- nucleotides do not disrupt an ORF. This simplifies the simula- chased from Coriell (Camden, NJ). I also amplified all 28 tion but does not affect the results, because the majority of exons from one chimpanzee (Pan troglodytes) and one orang- indels generated by mutations have small sizes (less than or utan (Pongo pygmaeus) and sequenced the insert DNAs after equal to nine nucleotides; Britten 2002). In the above geno- the PCR products were cloned into the pCR4TOPO vector mic data analyzed, 19% of the total 1019 indels are of sizes (Invitrogen). To trace the evolutionary origin of a large inser- that are multiples of three nucleotides. A simulation was then tion/deletion in exon 18, two segments (Figure 1, I and II) performed for 20,000 replications with the inferred ASPM of exon 18 were also amplified and directly sequenced from coding sequence of the common ancestor of humans and genomic DNAs of hyrax (Procavia capensis), sea lion (Zalophus chimpanzees. Under no functional constraints, the substitu- californianus), seal (Phoca vitulina), wolverine (Gulo gulo), fox tion rate is identical to the mutation rate and mutations are (Alopex lagopus), dog (Canis familiaris), bear (Ursus maritimus), assumed to be random. An ORF is interrupted when an indel cat (Felis catus), pig (Sus scrofa), cow (Bos taurus), whale (Ba- of a size that is not a multiple of three nucleotides or a non- laena mysticetus), rhesus monkey (Macaca mulatta), owl monkey sense point mutation occurs. I thus determined the t1/2 for (Aotus trivirgatus), and hamster (Cricetulus griseus). ASPM, or the time required for an intact ORF to be interrupted Data analysis: The dN/dS ratios (Li 1997; Nei and Kumar in half of the simulation replications. Under the above parame- ϭ 2000) in the human, chimpanzee, and orangutan branches ters, t1/2 0.48 MY. When point and indel mutation rates are ϭ of the ASPM gene tree (Figure 2) were estimated using the both halved, t1/2 0.97 MY. The probability that ASPM retains 1 T/t 1/2 maximum-likelihood method (Yang 1997), under the model its ORF after T MY of neutral evolution is ( ⁄2) . This estima- of unequal codon frequencies (CodonFreq ϭ 3) and unequal tion of the rate of pseudogenization is conservative, because rates of transitions and transversions. This model fits the data other deleterious events such as insertions of transposable significantly better than those with fixed codon frequencies elements and null mutations at promoter regions and splicing (CodonFreq ϭ 2) or equal rates of transitions and transver- sites are not considered here. The computer program for the ϭ sions. The hypothesis of dN/dS 1 for a given branch and the simulation is from Zhang and Webb (2003). hypothesis of equal dN/dS ratios between two branches were tested using the likelihood-ratio test, as well as a nonlikelihood method based on inferred ancestral sequences (Zhang et al. RESULTS 1997, 1998). The ASPM sequence for the common ancestor of humans and chimpanzees was estimated using the Bayesian Elevation of dN/dS in the human ASPM lineage: Hu- method (Yang 1997; Zhang and Nei 1997). Population ge- man ASPM has 28 coding exons, spanning 62 kb in netic analysis was conducted using DnaSP3.99 (Rozas and Rozas 1999). Tajima’s (1989), Fu and Li’s (1993), and Fay chromosome 1p31 and encoding a huge protein of 3477 and Wu’s (2000) tests were conducted using coalescent simula- amino acids (Figure 1). I determined the entire coding tions under the assumption of no recombination across the sequences of ASPM from one human, one chimpanzee, gene. This assumption makes the tests more conservative. and one orangutan, and compared them in the phyloge- When recombination is considered, the P values become netic tree of the three species (Figure 2). The orangutan lower in Tajima’s and Fu and Li’s tests, but remain virtually sequence is used as the outgroup for humans and chim- the same in Fay and Wu’s test. The recombination rate in the ASPM locus was estimated from Kong et al. (2002) to be 1.8 panzees so that nucleotide substitutions on the human cM/106 nucleotides. Since the coding sequences analyzed here and chimpanzee lineages can be separated. I did not span 62 ϫ 103 nucleotides of the genome, the recombination sequence the gorilla because the gorilla sequence may Ϫ rate for the sequence is r ϭ 1.8 ϫ 10 2 ϫ 0.062 ϭ 1.1 ϫ not be appropriate as the outgroup due to incomplete Ϫ3 10 recombination/generation.