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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.

SUBMITTER: Hollox EJ

PROVIDER: S-EPMC1180683 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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

Hollox E J EJ Armour J A L JA Barber J C K JC

American journal of human genetics 20030812 3

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 d ...[more]

PMID: 12916016

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Project description:IntroductionIt has been proposed that the copy number (CN) variation (CNV) in β-defensin genes (DEFB) on human chromosome 8p23 determines phenotypic differences in inflammatory diseases. However, no method for accurate and easy DEFB CN quantification is yet available.ObjectiveDroplet digital polymerase chain reaction (ddPCR) is a novel method for CNV quantification and has been used for genes such as CCL4L, CCL3L1, AMY1, and HER2. However, to date, no ddPCR assay has been available for DEFB CN determination. In the present study, we aimed to develop and evaluate such a ddPCR assay.MethodsThe assay was designed using DEFB4 and RPP30 as the target and the reference gene, respectively. To evaluate the assay, 283 DNA samples with known CNs previously determined using the multiple ligation-dependent probe amplification (MLPA) method, the current gold standard, were used as standards. To discover the optimal DNA template amount, we tested 80 to 2.5 ng DNA by a serial of one to two dilutions of eight samples. To evaluate the reproducibility of the assay, 31 samples were repeated to calculate the intra- and inter-assay variations. To further validate the reliability of the assay, the CNs of all 283 samples were determined using ddPCR. To compare results with those using quantitative PCR (qPCR), DEFB CNs for 48 samples were determined using qPCR with the same primers and probes.ResultsIn a one-dimensional plot, the positive and negative droplets were clearly separated in both DEFB4 and RPP30 detection channels. In a two-dimensional plot, four populations of droplets were observed. The 20 ng template DNA proved optimal, with either high (80 ng) or low (10, 5, 2.5 ng) template input leading to ambiguous or inaccurate results. For the 31 standard samples, DEFB CNs were accurately determined with small intra- and inter-assay variations (coefficient of variation < 0.04 for both). In the validation cohort, ddPCR provided the correct CN for all 283 samples with high confidence. qPCR measurements for the 48 samples produced noisy data with high uncertainty and low accuracy.ConclusionsddPCR is an accurate, reproducible, easy-to-use, cheap, high-throughput method for DEFB CN determination. ddPCR could be applied to DEFB CN quantification in large-scale case-control studies.

Dataset Information

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

Publications

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

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