Biason-Lauber, A.

Boehm, B.

Brun, T.

Gauthier, B.

Lang-Muritano, M.

Schoenle, E.

Wollheim, C.

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Diabetologia (2005) 48: 2185–2186DOI 10.1007/s00125-005-1925-xLETTERA. Biason-Lauber . B. Boehm . M. Lang-Muritano . B. R. Gauthier . T. Brun .C. B. Wollheim . E. J. SchoenleReply to comment on: Biason-Lauber A, Boehm B, Lang-MuritanoM et al. (2005) Association of childhood type 1 diabetes mellituswith a variant of PAX4: possible link to beta cell regenerativecapacity. Diabetologia 48:900–905Received: 6 July 2005 / Accepted: 22 July 2005 / Published online: 26 August 2005# Springer-Verlag 2005Abbreviations HWE: Hardy–Weinberg equilibriumTo the Editor: Some concerns have been raised that theanalytical methods we used for genotyping may be responsiblefor the striking differences observed in the distribution of thegenotypes of the PAX4 A1168C SNP, which deviated fromHardy–Weinberg equilibrium (HWE). In our certifiedlaboratory (ISO/IEC 17025, accreditation STS286) bothgenotypingmethods used (RFLP and allelic discrimination)were validated as described below.Pre-analytical phase To check the influence of samplepreparation (DNA extraction), transport and storage on theresults, DNA extractions were performed on ten EDTAblood samples. DNA extraction was performed immedi-ately after collection, 48 h after storage at room temperaturein the dark (mimicking the average shipment time), andfrom blood spots on filter paper. All extractions werefrozen, kept at −20°C for 7 days and then thawed beforeanalysis. The DNA extractions were performed as de-scribed previously [1].Analytical phase (1) Precision. To check reproducibility,we performed ten analyses of the ten EDTA blood sampleson the same day. We also assessed day-to-day precision byperforming a single analysis of each of the ten samples onfive different days. Laboratory precision was checked by asingle analysis of each of the ten samples by two differentoperators. (2) Accuracy. Five DNA samples, received froman associated laboratory, with known genotype (unknownto us) were each assayed on the same day. (3) Re-finding.Although the results are qualitative, in order to excludeartefacts due to DNA concentration, ten samples werediluted 1:5, 1:10 and 1:100 and analysed on the same day.Linearity and detection limits were not tested in this qual-itative assay.Our acceptance criterion was set at <5% discrepancy. Incase of a discrepancy either within one of the methods orbetween methods the discordant samples were analysed bysequencing of PCR‐amplified exonic fragments of exon 9.As a reference, three DNAs representing the three geno-types (A/A, A/C and C/C) were used in every series. Thereference sequences were CH236947.1 andAF043978. Thediscrepancy between the results of RFLP and sequencingwas 3%, whereas that between allelic discrimination andsequencing was 2%.All the case–control study samples were analysed byboth methods. As an internal control the same three sampleswith genotype confirmed by sequencing for the threevariants (A/A, A/C and C/C) plus a blank (no DNA) wereused in every series. To check the validity of the method, were-analysed the same ten samples per population everyfourth series (every 30×4=120 analyses for RFLP, and83×4=332 analyses for allelic discrimination). Since thebatches of reagents are sufficient for 200 and 600 RFLP andallelic discrimination reactions, respectively, this procedureA. Biason-Lauber . M. Lang-Muritano . E. J. SchoenleUniversity Children’s Hospital,Zurich, SwitzerlandB. BoehmUlm University,Ulm, GermanyB. R. Gauthier . T. Brun . C. B. WollheimUniversity Medical Centre,Geneva, SwitzerlandA. Biason-Lauber (*)Department of Paediatric Endocrinology/Diabetology,University Children’s Hospital,Steinvwiesstrasse 75,8032 Zurich, Switzerlande-mail: Anna.Lauber@kispi.unizh.chTel.: +41-1-2667623Fax: +41-1-2667169evaluates the influence of batch differences on the results.The discrepancy rate was comparable to that found in thevalidation series (2.9% for RFLP and 2.1% for allelicdiscrimination).Post-analytical phase (interpretation) To decrease the pos-sibility of biased interpretation of the results, we used twoapproaches. Firstly, all samples were analysed in a blindfashion—the samples were coded by numbers only. Thecode was revealed after completion of the analysis of theentire population (Swiss or German). For the Germanpopulation, operator and code-breaker were two differentindividuals. Secondly, to minimise congruency errors (cor-respondence case-result) due to data transfer errors (e.g.errors in the ordering of data in the Excel tables), the twopopulations were analysed separately and the results werelisted in separate tables. The chance that such errors occurrepeatedly and still generate the same trend in distributionshould be very low.We realised that although we found an associationbetween PAX4 variants and type 1 diabetes, the data are notin HWE. Of particular concern were the ‘excess heterozy-gotes’ in the control group of both populations. Sincedeviation from HWE in this case is most likely caused bygenotyping errors, we decided to address this issue using athird genotyping method, namely, sequencing (in additionto RFLP and allele discrimination, as described above andin [1]) to confirm our results. Sequencing produced similarresults, suggesting that the genotyping was correct. Themost probable explanation for this phenomenon is that thepolymorphism represents a heterozygote advantage, one ofthe known causes of deviation from HWE (together withoutbreeding, mutations, gene migration, genetic drift andnon-random mating). This is not an isolated phenomenonas it has been previously described and discussed [2–6].That the A/C genotype might confer a functional advantageis also confirmed by our expression studies. Althoughrather unlikely, there is always the possibility that a crypticpopulation stratification (a systematic difference in ances-try rather than association of genes with disease) mightinfluence our case–control data and that the correlationbetween type 1 diabetes and PAX4 observed in our popu-lations, although representative of the average populationsof these countries, might not be reproduced in other groups.Similarly, PAX4 variants have been linked to type 2 dia-betes in some ethnic groups but not in others [7, 8]. Thus,as underlined by A. Paterson, it is important to investigateother populations [9]. In this light, the data by Maier et al.[10], which are based on family studies, are very in-teresting. We are therefore currently studying other pop-ulations (Italian patients and control subjects) to find outwhether the ‘Swiss-German’ variant correlation will beconfirmed.Although diabetes remains a multifactorial disease andcan by no means be ascribed to a variation in a single gene,the role of PAX4 in the regeneration of beta cells issupported by our work [11] and indicates a link, at least incertain populations, between PAX4 defects and defects inthe regeneration of beta cells. Further studies, such as thoseoutlined by M. Tiedge in his commentary on our study[12], are necessary and are currently underway to identifyother components of this pathophysiological network.References1. Biason-Lauber A, Boehm B, Lang-Muritano M et al (2005)Association of childhood type 1 diabetes mellitus with a variantof PAX4: possible link to beta cell regenerative capacity.Diabetologia 48:900–9052. Rousset F, Raymond M (1995) Testing heterozygote excess anddeficiency. Genetics 140:1413–14193. Weitkamp LR, Tackels DC, Hunter AG, Holmes LB, SchwartzCE (1998) Heterozygote advantage of the MTHFR gene inpatients with neural-tube defect and their relatives. Lancet351:1554–15554. Nelson GW, Martin MP, Glasman D, Wade J, Trowsdale J,Carrington M (2004) Cutting edge: heterozygote advantage inautoimmune disease: hierarchy of protection/susceptibilityconferred by HLA and killer Ig-like receptor combination inpsoriatric arthritis. J Immunol 173:4273–42765. Balloux F (2004) Heterozygote excess in small populations andthe heterozygote-excess effective population size. Evolution IntJ Org Evolution 58:1891–19006. Correa PA, Gomez LM, Cadena J, Anaya JM (2005) Autoim-munity and tuberculosis: opposite association with TNF poly-morphsim. J Rheumatol 32:219–2247. Shimajiri Y, Sanke T, Furuta H et al (2001) A missensemutation of Pax4 gene (R121W) is associated with type 2diabetes in Japanese. Diabetes 50:2864–28698. Dupont S, Vionnet N, Chevre JC et al No evidence of linkageor diabetes-associated mutations in the transcription factorsBETA2/NEUROD1 and PAX4 in Type II diabetes in France.Diabetologia 42:480–4849. Paterson A (2005) Comment to Biason-Lauber A, Boehm B,Lang-Muritano M et al (2005) Association of childhood type 1diabetes mellitus with a variant of PAX4: possible link to betacell regenerative capacity. Diabetologia DOI 10.1007/s00125-005-1884-210. Maier LM, Cooper JD, Walker N, Smyth DJ, Todd JA (2005)Comment to Biason-Lauber A, Boehm B, Lang-Muritano M etal (2005) Association of childhood type 1 diabetes mellituswith a variant of PAX4: possible link to beta cell regenerativecapacity. Diabetologia DOI 10.1007/s00125-005-1906-011. Brun T, Franklin I, St-Onge L et al (2004) The diabetes-linkedtranscription factor PAX4 promotes beta-cell proliferation andsurvival in rat and human islets. J Cell Biol 167:1123–113512. Tiedge M (2005) Beta cell regeneration meets autoimmunity.Diabetologia 48:803–8042186

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Reply to comment on: Biason-Lauber A, Boehm B, Lang-Muritano M et al. (2005) Association of childhood type 1 diabetes mellitus with a variant of PAX4: possible link to beta cell regenerative capacity. Diabetologia 48:900-905

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