Global analysis of ATM polymorphism reveals significant functional constraint

Abstract

ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%-100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure.

Figure 1

Genotype data for 17 polymorphic sites in ATM, obtained for 91 representatives from seven major human populations. An asterisk (*) indicates that the polymorphism was not included in the haplotype analysis, because of recombination or recurrent mutation. White circles represent the homozygous ancestral allele, black circles the homozygous derived allele, and gray circles the heterozygotes. The nucleotide positions of the 17 polymorphic sites, in relation to the cDNA sequence (GenBank accession number U82828), are given at the top.

Figure 2

Phylogenetic relationship among seven ATM haplotypes. A chimpanzee sequence was used to determine the root of the phylogeny (H1). The circles each represent one haplotype, the area of the circle is proportional to the number of chromosomes observed with that haplotype, and the colors represent seven distinct geographic regions. The number of arrows indicates the number of changes required to generate the next related haplotype. Missense polymorphisms are indicated in parentheses. The number of observations are summarized in the table at the bottom of the figure. The haplotypes were defined by the nine congruent sites observed in ATM, including eight single-base-pair changes and one deletion. The nucleotide positions and the base pair changes defining the haplotypes are 378T→A, 657T→C, IVS8+38T→C, IVS9+25T→G, IVS17-56G→A, IVS24-9delT, IVS35-68T→G, 5557G→A, IVS62-55T→C, and 12563T→G where variation corresponding to amino acid changes is underlined. There are 133,342 bp between the first and the last markers. Haplotypes are defined as follows (the base pair change defining a new haplotype is underlined)—H1: TTTTATTGTT; H2: TTTTGTTGTT; H3: TTTTG(delT)TATT; H4: TTTTATTGCG; H5: ATTGATGGCG; H6: ACTGATGGCG; H7: ATCGATGGCG.

Figure 3

Distribution of coding-sequence polymorphisms in ATM and differences between chimpanzee and human. A, Location of polymorphisms and coding-sequence differences in chimpanzee compared to human. B, Location and frequency of polymorphisms observed in human ATM. The horizontal bar represents amino acid sequence of ATM from 1 to 3056. Colored and shaded regions of the amino acid sequence represent possible functional domains of the molecule: p53 binding (Khanna et al. 1998), leucine zipper (Morgan et al. 1997), homology to S. pombe Rad3p (Bentley et al. 1996) and S. cerevisiae Tel1p (Greenwell et al. ; Morrow et al. 1995), and the serine kinase domain (Zakian 1995). Ball-and-stick figures along the amino acid sequence represent amino acid positions. Circles are mapped at positions that are polymorphic either within the 93 human individuals (with the number of individuals adjacent to each circle) or within the single chimpanzee individual; diamonds represent homozygous differences between human and chimpanzee. Black circles or diamonds represent missense polymorphisms, and white circles or diamonds represent silent changes.

Figure 4

Nucleotide diversity in coding and noncoding regions of 15 autosomal and 3 Y-chromosome genes. Nucleotide diversity, or heterozygosity, was calculated as described in the Subjects, Material, and Methods section. Autosomal genes are shown in black and gray, and Y-chromosome genes are shown in red and pink.