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  • Allelic recombination between distinct genomic locations generates copy number diversity in human �-defensins Suhaili Abu Bakara, Edward J. Holloxb, and John A. L. Armoura,1

    aInstitute of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, United Kingdom; and bDepartment of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom

    Edited by Walter Bodmer, John Radcliffe Hospital, Oxford, United Kingdom, and approved November 13, 2008 (received for review September 11, 2008)

    �-Defensins are small secreted antimicrobial and signaling pep- tides involved in the innate immune response of vertebrates. In humans, a cluster of at least 7 of these genes shows extensive copy number variation, with a diploid copy number commonly ranging between 2 and 7. Using a genetic mapping approach, we show that this cluster is at not 1 but 2 distinct genomic loci �5 Mb apart on chromosome band 8p23.1, contradicting the most recent genome assembly. We also demonstrate that the predominant mechanism of change in �-defensin copy number is simple allelic recombina- tion occurring in the interval between the 2 distinct genomic loci for these genes. In 416 meiotic transmissions, we observe 3 events creating a haplotype copy number not found in the parent, equiv- alent to a germ-line rate of copy number change of �0.7% per gamete. This places it among the fastest-changing copy number variants currently known.

    Humans differ in diploid DNA dosage of certain regions. Theintroduction of new technologies has allowed a more accurate appreciation of the true scale and frequency of this dosage varia- tion, or copy number variation (CNV), in the human genome (1–4). It is clear that it contributes to variation in gene expression (5) and that it is associated with variable phenotypes (6), including infec- tious and autoimmune disease (7–9). Although there have been interesting recent studies on the mechanisms by which copy number may be altered in the germ line (10–13), there are relatively few loci that have been studied in detail, and it is likely that mutation rates and mechanisms will differ between individual loci. The CNV in human �-defensin genes involves a cluster of at least 7 �-defensins in 8p23.1, including genes encoding proteins known to have anti- microbial properties (14) but which also have other functions and effects (15, 16). For most of these genes there is little detailed information about function and expression (14), and the �-defensin CNV forms part of a complex spectrum of population and evolu- tionary dynamics in this region of the genome (17). Nearly all individuals have between 2 and 7 copies of this cluster per diploid genome (18–21), but some individuals can have up to 12 copies, with the highest-copy-number haplotypes forming the basis of the cytogenetic 8p23.1 euchromatic variant (18, 22). In the more frequent copy number range, low �-defensin copy number has been reported to be associated with Crohn’s disease of the colon (23) and high copy number with predisposition to psoriasis (20). This common variation may be involved in predisposition to other inflammatory disorders, but progress in these association studies has been hampered by the difficulty of measuring copy number accurately for large numbers of samples (24, 25).

    This region contains another large-scale structural polymor- phism: a common inversion that involves nearly the whole chro- mosome band (26, 27), which is apparently mediated by recombi- nation between clusters of repeats at the flanking proximal (REPP) and distal (REPD) regions (Fig. 1). REPP and REPD contain not only olfactory receptor gene repeats but also complex and poly- morphic clusters of FAM90A genes (28). Approximately one- quarter of Europeans and one-third of Japanese are heterozygous for this inversion; in these heterozygotes unequal recombination

    can lead to the formation of the pathological rearrangement inv dup(8p) or its reciprocal product, �der (8) (26, 29).

    The combination of frequent structural polymorphism involving extensive repeat-rich regions of unknown variable size makes the sequence of the REPD and REPP regions extremely difficult to assemble (30). Because of the large size of the repeats and the multiple individuals used to generate BAC libraries used in genome assemblies, even long Sanger sequencing reads and deep clone coverage have not, as yet, resolved a gap at this region in the human genome assembly. The REPD and REPP regions are very similar to other olfactory repeat regions, which are themselves copy number variable (31). This makes selecting clones for cytogenetic probes and accurately interpreting results difficult because of variable hybridization intensities against different olfactory repeat regions and extensive cross-hybridization. An accurate representa- tion of the sequence of this region may be resolved only by constructing several alternative allelic assemblies and determining the frequency of those allelic assemblies in the population. It is likely that the uncertainty surrounding this region is responsible for misinterpretation of cytogenetic signals in some studies (32, 33).

    We decided to use a genetic approach to investigate the structure and mutation processes at the �-defensin CNV, which does not a priori assume correct physical mapping of the region. Little is known about the processes generating and maintaining CNV; most new mutation is assumed, and in some cases has been demon- strated, to occur by nonallelic homologous recombination between flanking repeats generating reciprocal products with a loss and a gain of a repeat, respectively. Given the functional consequences of this region and its relevance to disease, it is clearly important that these questions are addressed.

    Results To examine mutation processes at the �-defensin CNV, we ana- lyzed inheritance of the region in pedigrees for de novo mutation or recombination events. We determined copy numbers of the variable �-defensin CNV in Centre d’Etude du Polymorphisme Humain (CEPH) family members using the paralogue ratio test (PRT), combined with analysis of variant ratios at microsatellites EPEV-1 (18) and EPEV-3, and the multiallelic length polymor- phism including the indel rs5889219. PRT is a development of the multiplex comparative PCR approach that uses a single primer pair to amplify both test and reference loci, leading to more accurate and robust copy number determination (24). Ratios of products from

    Author contributions: E.J.H. and J.A.L.A. designed research; S.A.B., E.J.H., and J.A.L.A. performed research; E.J.H. and J.A.L.A. analyzed data; and S.A.B., E.J.H., and J.A.L.A. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission.

    1To whom correspondence should be addressed. E-mail:

    This article contains supporting information online at 0809073106/DCSupplemental.

    © 2009 by The National Academy of Sciences of the USA�cgi�doi�10.1073�pnas.0809073106 PNAS � January 20, 2009 � vol. 106 � no. 3 � 853–858

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  • multiallelic length polymorphisms could be used to confirm copy number measurements; for example, amplification of 3 variants with yields in the ratio 2:2:1 strongly suggests a copy number of 5 (see, for example, the rs5889219 profile of 133306 in Fig. 2). Although not fully informative (for example, a 6-copy sample might have variants in the ratio 2:1, compatible with any multiple of 3), these ratio-based measurement methods provided valuable addi- tional information by distinguishing different repeat identities in the analysis of segregation. By contrast, simple ‘‘copy-counting’’ meth- ods like PRT give unambiguous information on copy number but provide no information on the individual identities of the repeat units.

    We combined these approaches to establish the pattern of �-defensin transmission in 208 offspring from 26 CEPH families. Consistent with previous data (18–21), copy number among the parents of these 26 families varied between 2 and 7, with a modal copy number of 4 and a mean of 4.58. We observed that at least 24 offspring inherited recombinant haplotypes in which parental cop- ies of the �-defensin repeat had been reassorted. In many instances the haplotype copy number was unchanged, but in some cases a new copy number was found in the recombinant chromosome; in Fig. 2, for example, child 133306 receives a recombinant maternal chro- mosome carrying 3 repeat units, whereas children inheriting non- recombinant haplotypes show that the mother (133302) has 2 2-copy haplotypes. The data in Fig. 3 show that children 134106 and 134109 have each received different (reciprocal) recombinant maternal haplotypes, but with no change in the haplotype copy number. Most unexpectedly, mapping the positions of the crossover breakpoints using CEPH segregation data showed that these re- combinations cannot all simply be occurring within a coherent block of �-defensin tandem repeats; the breakpoint of the maternal crossover in 133306, for example, is located proximal to rs2001329 at position chr8:11,024,269 on the March 2006 Genome Assembly, �3 Mb proximal to the assembly position of the �-defensin repeats

    (Fig. 4). In some transmissions, for example, the paternal chromo- some of 1329208 [see supporting information (SI) Fig. S1], where parental repeats share m