Extensive normal copy number variation of a beta-defensin antimicrobial-gene cluster

Abstract

Using a combination of multiplex amplifiable probe hybridization and semiquantitative fluorescence in situ hybridization (SQ-FISH), we analyzed DNA copy number variation across chromosome band 8p23.1, a region that is frequently involved in chromosomal rearrangements. We show that a cluster of at least three antimicrobial beta-defensin genes (DEFB4, DEFB103, and DEFB104) at 8p23.1 are polymorphic in copy number, with a repeat unit >/=240 kb long. Individuals have 2-12 copies of this repeat per diploid genome. By segregation, microsatellite dosage, and SQ-FISH chromosomal signal intensity ratio analyses, we deduce that individual chromosomes can have one to eight copies of this repeat unit. Chromosomes with seven or eight copies of this repeat unit are identifiable by cytogenetic analysis as a previously described 8p23.1 euchromatic variant. Analysis of RNA from different individuals by semiquantitative reverse-transcriptase polymerase chain reaction shows a significant correlation between genomic copy number of DEFB4 and levels of its messenger RNA (mRNA) transcript. The peptides encoded by these genes are potent antimicrobial agents, especially effective against clinically important pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, and DEFB4 has been shown to act as a cytokine linking the innate and adaptive immune responses. Therefore, a copy number polymorphism involving these genes, which is reflected in mRNA expression levels, is likely to have important consequences for immune system function.

Figure  1

MAPH probe locations on 8p23.1. Positions of the MAPH probes (A–N) used to screen 8p23.1 for copy number variation are shown by arrows, together with the ORRs named “REPP” and “REPD” by Giglio et al. (2001). The area identified as involved in the copy number variation is shown in detail and is represented twice in this genome assembly (November 2002 assembly; for details, see the University of California–Santa Cruz [UCSC] Genome Bioinformatics Web site). The probe marked with an asterisk (*) showed increased dosage in 8p23.1 EVs but maps to ORR sequences and was not included in the main MAPH probe set. RefSeq = reference sequence.

Figure  2

MAPH analysis of normal control individual and 8p23.1 EV carrier. A MAPH chromatogram shows the analysis of an EV carrier (bottom), with 12 copies of the 8p23.1 repeat unit, and a control DNA sample (top), with 3 copies of the 8p23.1 repeat unit. The X-axis represents probe length (in bp), and the Y-axis represents relative fluorescence units. Each peak represents a different amplifiable probe separated by length. The three probes (F–H) measuring β-defensin–cluster copy number are indicated, together with a probe (E) mapping to DEFA1. Other peaks represent other probes, most of which (47 of the remaining 57) map to single-copy subtelomeric regions and have a copy number of two per diploid genome. The areas of the polymorphic probe peaks (F–H) in the control sample (three copies) are similar to, but slightly larger than, the subtelomeric probe peaks (two copies).

Figure  3

SQ-FISH of 8p23.1 EV (left) and normal (right) homologs in individual II:1 of family 3, using 8p23.1 BAC 51D11 (red) and D8Z2 centromere probe (green).

Figure  4

MAPH analysis of 8p23.1 copy number in three families. Pedigrees of families 1–3 are annotated with MAPH-determined copy number (see also table 2). Half-blackened symbols indicate EV carriers. N = chromosomally normal; nt = not tested.

Figure  5

DEFB4 transcript levels versus β-defensin–cluster DNA copy number for a series of seven lymphoblastoid cell lines. DEFB4 transcript was measured in triplicate by using duplex RT-PCR with TBP as a control transcript. Mean ratio (transformed to log₁₀) ± SEM of DEFB4:TBP intensity ratios is shown on the Y-axis, and MAPH copy number is shown (mean of five tests ± SEM) on the X-axis. There is a significant correlation between DEFB4 transcript levels and β-defensin–cluster copy number (r²=0.5; P<.05). A similar pattern of variation in DEFB4 expression level was also obtained, using G3PD as a control transcript (data not shown).