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...y number (5), and case–control studies have provided the clearest indications to date of the functional consequences of this CNV. Given the functions of beta-defensins as antimicrobials and cytokines =-=(6,7)-=-, investigators have tested for association between beta-defensin variation and inflammatory disease, including psoriasis (8) and Crohn’s disease (9,10). In 2006, ∗To whom correspondence should be add...

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...ibution of betadefensin copy number classes between the controls of European origin typed in this study, by Fellermann et al., by Bentley et al. and of the European controls typed by McCarroll et al. =-=(22)-=-. The distribution of copy number classes found in this study, or by Fellermann et al., shows no significant discrepancy with that found by McCarroll et al.; however, although it remains possible that...

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...f beta-defensins as antimicrobials and cytokines (6,7), investigators have tested for association between beta-defensin variation and inflammatory disease, including psoriasis (8) and Crohn’s disease =-=(9,10)-=-. In 2006, ∗To whom correspondence should be addressed. Tel: +44 1158230308; Fax: +44 1158230313; Email: john.armour@nottingham.ac.uk # The Author 2010. Published by Oxford University Press. All right...

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...V. Given the functions of beta-defensins as antimicrobials and cytokines (6,7), investigators have tested for association between beta-defensin variation and inflammatory disease, including psoriasis =-=(8)-=- and Crohn’s disease (9,10). In 2006, ∗To whom correspondence should be addressed. Tel: +44 1158230308; Fax: +44 1158230313; Email: john.armour@nottingham.ac.uk # The Author 2010. Published by Oxford ...

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...disease would come from an analysis that explicitly accounted for the measurement error in the evaluation of evidence for association, such as that formulated in the CNVtools package of Barnes et al. =-=(21)-=-. We attempted to apply these improved methods of analysis to our PRT data, but were unable to obtain convergence in the clustering phase of the analysis; we speculate that this failure comes from a c...

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...eral hundred samples could easily generate spurious associations attributable either directly to differential error or to differential removal of samples failing to satisfy a typing quality threshold =-=(17)-=-. Given that the highest copy numbers are intrinsically the most error-prone, even small differences of quality in the underlying CNV measurements could lead to an artefactual shift in the copy number...

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...y number (5), and case–control studies have provided the clearest indications to date of the functional consequences of this CNV. Given the functions of beta-defensins as antimicrobials and cytokines =-=(6,7)-=-, investigators have tested for association between beta-defensin variation and inflammatory disease, including psoriasis (8) and Crohn’s disease (9,10). In 2006, ∗To whom correspondence should be add...

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... for reporting spurious associations with CNVs, especially multiallelic CNVs. The WTCCC study also failed to replicate the reported association of rheumatoid arthritis with CCL3L1/ CCL4L1 copy number =-=(24)-=-, which (like the reported associations of Crohn’s disease with beta-defensin copy number) relied on copy number determination by real-time PCR, unsupported by independent methods. The studies of both...

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...nic at the Western General Hospital, Edinburgh, Scotland, which is a tertiary referral centre for IBD in South-East Scotland. The diagnosis of Crohn’s disease adhered to the criteria of Lennard-Jones =-=(26)-=-. Age at diagnosis and disease phenotype (location and behaviour) were classified according to the Montreal classification (27). Written informed consent was obtained from all patients. Healthy contro...

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...s, including DEFB4, DEFB103–107 and SPAG11, but excluding the neighbouring DEFB1 gene. There is a correlation between copy number and beta-defensin gene expression at the mRNA level in isolated cells =-=(1)-=-, though a recent study found that beta-defensin 2 protein production from ex vivo-cultured colonic biopsies was correlated with local inflammation, but not with DEFB4 copy number (5), and case–contro...

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...pair to amplify both test and reference amplicons simultaneously, so that the potential error arising from the different amplification efficiencies of distinct test and reference amplicons is reduced =-=(18)-=-. Field et al. showed an apparently highly significant association between CCL3L1 copy number and type I diabetes using real-time PCR data. However, these same data showed a significant deviation from...

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...he diagnosis of Crohn’s disease adhered to the criteria of Lennard-Jones (26). Age at diagnosis and disease phenotype (location and behaviour) were classified according to the Montreal classification =-=(27)-=-. Written informed consent was obtained from all patients. Healthy controls were either unrelated spouses/friends of IBD patients or anonymized blood samples obtained from the Scottish Blood Transfusi...

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...ber, using real-time PCR measurement in 466 cases and 329 controls. Measuring copy number accurately poses particular challenges for case–control association studies, especially in multi-allelic CNVs =-=(11)-=-, such that distinguishing (for example) five copies of the beta-defensin segmental duplication reproducibly from six copies is not easily achievable using standard methods. The unresolved influence o...

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... variable repeat unit have clearly shown that the entire repeat unit varies coordinately, so that the measurement of any element in the repeat constitutes an effective copy number assay for the whole =-=(1,19,20)-=-. Our ‘triplex’ assay contains three components: a ‘PRT’ (PRT107A) and rs5889219 multi-allelic indel measure already described (19) together with a newly modified PRT assay (‘HSPD21’) based on the sam...

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...eral failure of real-time PCR methods to resolve copy number clusters clearly for n. 2 at the CCL3L1 copy number variant (13,14,16). This observation is consistent with the experience of Perne et al. =-=(25)-=-, who concluded that real-time PCR was the least consistent of the three methods they tested for beta-defensin copy number measurement. Very generally, whatever copy number measurement is used, in ord...

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...t al. explicitly compared the data from the same samples (several thousand cases and controls in a study of type I diabetes), using either real-time PCR or assays using the paralogue ratio test (PRT) =-=(14)-=-. PRT uses a single primer pair to amplify both test and reference amplicons simultaneously, so that the potential error arising from the different amplification efficiencies of distinct test and refe...

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...nd Oncology Subcommittee of the Lothian Local Research Ethics Committee approved the study protocol (LREC 2000/ 4/192). DNA was extracted by either a modified salting out technique as used previously =-=(28)-=- or by using a Nucleon blood DNA extraction kit according to the manufacturer’s protocol. Of the 435 (total) Crohn’s disease samples typed in this study, 418 were also included in the WTCCC (29). Figu...

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...ndon’) DNA samples designated as extracted at the ‘London’ Centre were white European patients with Crohn’s disease recruited from specialist IBD clinics in London and Newcastle as reported elsewhere =-=(30)-=- after informed consent and ethical review (REC 05/Q0502/127). DNA was extracted from 10 ml of venous blood by salt/chloroform extraction (30). Of the 666 samples used in this study, 491 were also inc...

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...otorgene (Corbett Research, now Qiagen, UK). Taqman primers and probes were obtained (Applied Biosystems, UK) as 20× mixes. ALB was used as a reference gene as it has a single copy per haploid genome =-=(31)-=-. Cycling conditions were: initial hold at 958C for 3 min followed by 45 cycles of: 958C for 20 s, 608C for 30 s, 728C for 30 s. Primer sequences used: DEFB4 forward primer: TGTGGCTGATGCGG ATTCA, DEFB...

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...n isolated cells (1), though a recent study found that beta-defensin 2 protein production from ex vivo-cultured colonic biopsies was correlated with local inflammation, but not with DEFB4 copy number =-=(5)-=-, and case–control studies have provided the clearest indications to date of the functional consequences of this CNV. Given the functions of beta-defensins as antimicrobials and cytokines (6,7), inves...

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...f beta-defensins as antimicrobials and cytokines (6,7), investigators have tested for association between beta-defensin variation and inflammatory disease, including psoriasis (8) and Crohn’s disease =-=(9,10)-=-. In 2006, ∗To whom correspondence should be addressed. Tel: +44 1158230308; Fax: +44 1158230313; Email: john.armour@nottingham.ac.uk # The Author 2010. Published by Oxford University Press. All right...

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...RT, carefully designed and implemented to address the difficulty of the CNV-counting problem, are only capable of making the necessary fine distinctions of dosage with some level of unavoidable error =-=(8,18,19)-=-. In this work, we combine PRT methods of CNV determination modified to reduce typing error with careful analysis of typing results to analyse beta-defensin copy number in more than 1000 cases of Croh...