Processes of copy-number change in human DNA: the dynamics of {alpha}-globin gene deletion - PubMed (original) (raw)

Processes of copy-number change in human DNA: the dynamics of {alpha}-globin gene deletion

Kwan-Wood G Lam et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Ectopic recombination between locally repeated DNA sequences is of fundamental importance in the evolution of gene families, generating copy-number variation in human DNA and often leading to pathological rearrangements. Despite its importance, little is known about the dynamics and processes of these unequal crossovers and the degree to which meiotic recombination plays a role in instability. We address this issue by using as a highly informative system the duplicated alpha-globin genes in which ectopic recombination can lead to gene deletions, often very prevalent in populations affected by malaria, as well as reduplications. Here we show that spontaneous deletions can be accessed directly in genomic DNA by using single-DNA-molecule methods. These deletions proved to be remarkably common in both blood and sperm. Somatic deletions arise by a strictly intrachromosomal pathway of homologous exchange that also operates in the germ line and can generate mutational mosaicism, whereas sperm deletions frequently involve recombinational interactions between homologous chromosomes that most likely occur at meiosis. Ectopic recombination frequencies show surprisingly little requirement for long, identical homology blocks shared by paralogous sequences, and exchanges can occur even between short regions of sequence identity. Finally, direct knowledge of germ-line deletion rates can give insights into the fitness of individuals with these alpha-globin gene deletions, providing a new approach to investigating historical levels of selection operating in human populations.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Detection of de novo deletions in the α-globin gene region. (A) The region analyzed, showing the α-globin genes and pseudogene, plus BamHI cleavage sites used for size fractionation of genomic DNA and nested PCR primers (arrows) used to amplify deletion molecules. Similar sequences are colored to highlight homologies, with levels of sequence divergence between each homology block and its paralog immediately to the right shown below. (B) Leftward and rightward deletions that can arise by ectopic exchange. SNP heterozygosities [white (haplotype A) and black (haplotype B) circles] make it possible to distinguish interchromosomal recombinants showing exchange of flanking markers (AB and BA) from intrachromosomal deletions (AA and BB). (C) Examples of detection of deletion molecules. Aliquots of one of the fractions of blood DNA (10.2- to 11.4-kb BamHI DNA fragments), each derived from 1.1 × 105 amplifiable haploid genomes and containing ≈0.5 remaining 14.1-kb progenitor DNA molecules, were amplified by nested PCR, and products were analyzed by agarose gel electrophoresis. M, λ DNA × HindIII. Some reactions show PCR products derived from deletion mutants, and some others show progenitor molecules. Differential amplification results in mutant molecules being substantially overamplified relative to the longer progenitor. (D) Cumulative frequencies of deletion molecules across size fractions of BamHI-digested sperm DNA, determined from a total of 90 −α3.7 and 19 −α4.2 deletion mutants recovered from 6.7 × 106 progenitor molecules. Size ranges covered by fractions are shown as gray bars. The control is a 10.0-kb genomic BamHI fragment matched in size to the −α4.2 deletion mutant.

Fig. 2.

Fig. 2.

α-globin gene deletions detected in sperm and blood DNA. (A) The region analyzed, as described for Fig. 1. (B) Structure of −α4.2 (Left) and −α3.7 (Right) deletions, with PSVs marked as rectangles. (i) Examples of typical deletions mapping to a single interval between PSVs. A total of 539 and 128 such exchanges were seen in sperm and blood DNA, respectively. (ii) Complex mutants with PSV switching (∗) near the site of ectopic exchange. Nine such −α4.2 mutants were identified in the sperm of man 2. The complex −α3.7 deletion was seen in three blood mutants from man 1 and two sperm mutants from man 2. (iii) A simple exchange accompanied by a microdeletion in a distal Alu repeat (red sequence lost) seen in one blood mutant. (C) Distribution of ectopic exchange points. Progenitor haplotypes have heterozygous SNPs marked as black and white circles. Each interval of sequence identity containing exchange breakpoints is marked by a tied horizontal line, in blue for −α4.2 deletions, with the number of exchanges seen in sperm and blood DNA indicated in black and red, respectively, above the tie (−, no mutants). Intra- and interchromosomal deletions are shown separately. Some SNPs are located within homology blocks and, depending on the allele, can break long regions of identity into smaller regions, creating differences between haplotypes in the intervals within which ectopic exchanges can be mapped. These mutants were recovered from 6.5 × 106 and 12.9 × 106 progenitor molecules from sperm and blood DNA, respectively, from man 1 and 6.7 × 106 and 7.1 × 106 molecules from man 2.

Fig. 3.

Fig. 3.

Distribution of ectopic exchange points within homology blocks. (A) The homologous regions analyzed, colored as described for Fig. 1 and with PSVs marked by lines below. SNPs in one haplotype that disrupt a region of perfect sequence identity in the other haplotype are marked in red. (B) Cumulative number of ectopic exchanges, per 106 haploid genomes, across homology blocks for all −α4.2 exchanges combined and for each type of −α3.7 exchange, with intrachromosomal sperm exchanges in man 1 shown separately. (C) Exchange frequencies per base pair estimated for each interval of sequence identity, with the best-fit logarithmic curve after excluding the outlying points (arrows). The circled points with a low rate correspond to the interval marked by an asterisk in A.

Fig. 4.

Fig. 4.

The effect of fitness on the incidence of −α deletion chromosomes in nonmalarial populations. The expected population incidence of deletions at mutation/selection equilibrium was estimated from the observed germ-line deletion rate of 42 × 10−6 per sperm, averaged over the two men analyzed and assumed to be the same in the female germ line. Frequencies were estimated at various fitness levels of −α/−α homozygotes and with different fitness levels _f_c of αα/−α carriers. The dashed line provides the estimated incidence of −α chromosomes in north Europeans, with 95% confidence intervals indicated in gray. See Materials and Methods for details.

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