Lack of Association of PAX4 Gene With Type 1 Diabetes in the Finnish and Hungarian Populations
http://www.100md.com
糖尿病学杂志 2005年第9期
1 Juvenile Diabetes Research Foundation Centre for Prevention of Type 1 Diabetes in Finland, Turku, Finland
2 Department of Virology, University of Turku, Turku, Finland
3 Department of Pediatrics, University of Oulu, Oulu, Finland
4 Department of Pediatrics, University of Pecs, Pecs, Hungary
5 Program of Developmental and Reproductive Biology, Biomedicum, University of Helsinki, Helsinki, Finland
6 Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland
7 Department of Pediatrics, University of Turku, Turku, Finland
8 Department of Pediatrics, Tampere University Hospital, Tampere, Finland
ABSTRACT
We aimed to assess the possible contribution of the PAX4 transcription factor gene to the genetic background of type 1 diabetes. We analyzed four coding polymorphisms of the PAX4 gene in 498 cases with type 1 diabetes and 825 control subjects from Finland and Hungary. All patients were diagnosed under the age of 15 years according to the World Health Organization criteria. All four PAX4 variants (three in exon 9 and one in exon 3) were genotyped using DNA sequencing. In addition, all Finnish subjects were typed for HLA DR-DQ, insulin gene (eC23) HphI, and CTLA4 CT60 polymorphisms. The +1,168 C/A coding variant of PAX4 was found to be polymorphic in both populations (P321H, rs712701). No difference was observed in the genotype frequencies between cases and control subjects, nor was any disease association detected when patients were stratified according to age at diagnosis, sex, HLA, insulin gene, or CTLA4 genotypes. Our data indicate that the +1,168 C/A variant of PAX4 gene does not play any essential role in genetic type 1 diabetes susceptibility. The strong coherence between the datasets of the two ethnic groups studied with highly contrasting disease incidence, socioeconomic characteristics, and profoundly diverse environment emphasizes the impact of this finding.
Type 1 diabetes is caused by immune-mediated destruction of the insulin-producing pancreatic -cells that results from a complex interplay between a polygenic background and environmental factors. In addition to the HLA complex, the insulin gene (INS), the cytotoxic T-celleCassociated protein 4 gene region (CTLA4), and the protein tyrosine phosphatase gene (PTPN22) are confirmed disease susceptibility loci (1,2). A number of other gene regions have also been found to show evidence of linkage or association with type 1 diabetes. The confirmation of true genetic effects in this complex disease remains a challenge for several reasons. One of the main hurdles is the modest disease predisposing effect of these loci (odds ratio [OR] < 2) that is further complicated by the genetic heterogeneity of the extensive patient cohorts that are required to ensure statistical power. As an example, recently two groups reported association of the small ubiquitin-like modifier 4 gene (SUMO4; OMIM 608829; 6q25) with type 1 diabetes (3,4). However, these findings were not confirmed in two large multiethnic family sets, indicating the complexity of identifying real underlying gene effects for type 1 diabetes (5,6).
The pancreatic -cell mass of an individual is tightly regulated according to insulin demand and reflects the balance between the rate of -cell replication and neogenesis and the rate of -cell apoptosis (7). During an autoimmune attack, the rate of -cell death and the capacity of -cell regeneration could be crucial factors in determining the progression of the imbalance in glucose homeostasis (8). It is known that a network of transcription factors provide genetic instructions for the differentiation and development of pancreatic -cells (9). Any alteration of this sophisticated mechanism could lead to reduced -cell mass or function and may even determine whether clinical diabetes develops.
Two recent reports have suggested that the gene encoding the pancreatic transcription factor PAX4 (OMIM 167413; 7q32) is associated with type 1 diabetes (10,11). In the current study, we aimed to assess this association in two populations with high and low incidence rates of type 1 diabetes. These populations also show conspicuous differences in terms of socioeconomic conditions, climatic, and other environmental factors.
RESEARCH DESIGN AND METHODS
All patients with type 1 diabetes were diagnosed at <15 years of age according to the World Health Organization criteria.
Finnish population series.
Children with type 1 diabetes were derived mainly from the Departments of Pediatrics at the universities in Turku, Helsinki (southern Finland), and Oulu (northern Finland) (n = 309, 50.5% males; mean age at diagnosis 8.4 ± 4.2 years). The control group comprised consecutive healthy infants born in the University Hospitals of Turku and Oulu who were randomly selected for this study (n = 535, 47.5% males).
Hungarian population series.
Patients were consecutively diagnosed in Baranya County (southern Hungary) over a 17-year period from 1980 to 1996 and recruited through the Hungarian Childhood Diabetes Registry (n = 189, 49.7% males; mean age at diagnosis 8.5 ± 4.8 years). Healthy schoolchildren born in the same geographical region and matched for sex and age were used as control subjects (n = 290, 50.7% males; mean age at sampling 10.6 ± 2.7 years).
Four single nucleotide polymorphisms (SNPs) in the PAX4 gene (OMIM 167413, 7q32) were analyzed using DNA sequencing (MegaBace1000 DNA Sequencer; GE Healthcare). Three nonsynonymous SNPs were located in exon 9 [rs712701 (P321H), rs2233583 (V330A), and rs2233584 (Ter333W)], whereas the rs2233578 SNP (R133W) was in exon 3. HLA typing was performed in all Finnish subjects using methods described earlier (12,13). The INS eC23 HphI SNP was typed with a similar oligonucleotide hybridization format (T probe 5'-Europium-CTGTCTCCCAGA-3'; A probe 5'-Terbium-CTGTCACCCAGA-3'). The CTLA4 CT60 SNP was genotyped using minisequencing (SnuPe kit; GE Healthcare). Primer sequences are available on request.
Statistical analysis.
For comparisons of genotype frequencies between the groups, 2 statistics with Yates’ correction were used. P values <0.05 were considered significant. The effect of PAX4 and the other studied genes was also examined by logistic regression analysis (SPSS for Windows, version 11.0.1; SPSS, Chicago, IL). Power calculations were done using standard methods.
RESULTS
The R133W variant in exon 3 (rs2233578) was analyzed in 46 healthy newborn infants and in 124 patients. One Finnish patient carried the arginine/tryptophan heterozygous genotype; all other subjects were arginine/arginine homozygous. At the V330A polymorphism in exon 9 (rs2233583), one child with type 1 diabetes from Finland carried the heterozygote genotype, whereas all other individuals (n = 1,322) were alanine/alanine homozygous. In addition, no variation was detected at the rs2233584 SNP (Ter333W) among the 1,323 study subjects.
The C/A variant at coding sequence position +1,168 is a nonsynonymous SNP (P321H; rs712701) that was found polymorphic. No differences in the genotype frequencies between cases and control subjects were observed in the Finnish and Hungarian series (Table 1). Accordingly, the comparison on the combined datasets showed no difference between patients and control subjects. The genotype distributions were in Hardy-Weinberg equilibrium both among cases and control subjects in the two series (P = 0.82 and 0.69, respectively, for the Finnish set; P = 0.50 and 0.96, respectively, for the Hungarian set). We stratified the Finnish patients according to age at diagnosis and sex and compared the PAX4 genotype frequencies with control subjects, but no differences were seen (Table 1). Moreover, to elucidate gene-gene interactions, we analyzed the distribution of the PAX4 genotypes in patients stratified according to the HLA DRB1-DQA1-DQB1, insulin gene (eC23) HphI, and CTLA4 CT60 genotypes and observed no differences (Table 1). The lack of association of PAX4 with type 1 diabetes was confirmed also by logistic regression analysis.
We compared the genotype and allele frequencies (rs712701 SNP) in the Finnish newborn series between northern and southern Finland, and no differences were detected (data not shown). Similarly, no regional differences were either observed in the genotype frequencies among the Finnish patients (data not shown, P = 0.69, df = 4). In contrast, the +1,168 genotype distributions showed a difference between the Finnish and Hungarian control series (P = 0.002) since the A allele was more frequent in the Finnish population (27.0 vs. 19.5% in Hungarians, P = 0.0008). As expected, genotype distributions were also different between the two populations among the patients affected by type 1 diabetes (P = 0.007).
DISCUSSION
The identification of novel type 1 diabetes genes is challenging since the disease is polygenic and the current mapping methods, such as linkage analysis, seem to have very low sensitivity, and association studies are extremely sensitive to population stratification biases. The situation is further complicated by linkage disequilibrium heterogeneity that exists between different ethnic groups (14). In addition, it is likely that there are several alternative disease pathways, and the effect of certain genetic variants is likely to be restricted to a particular molecular mechanism. Our previous studies suggest that the INS locus is associated with the emergence of insulin autoantibodies and could play a role in the development of insulin autoimmunity (15,16). In contrast, INS was not associated with multiple autoantibody positivity in children without insulin autoantibodies (16). As a consequence, new strategies for mapping of genes predisposing to type 1 diabetes are required using such cohorts, where the patients are well characterized for molecular markers such as -celleCspecific autoantibodies.
In this study, we attempted to reproduce the findings of Biason-Lauber et al. (10) on the association of the gene encoding the PAX4 transcription factor and type 1 diabetes. However, we were unable to confirm a role for the +1,168 polymorphism of PAX4 in disease pathogenesis in the two populations studied with profoundly different socioeconomic conditions, climate, and environment. Similarly, no effect of PAX4 could be seen in any patient subgroups analyzed according to age at diagnosis, sex, HLA, and CTLA4 CT60 genotypes. Since PAX4 is a pancreatic transcriptional regulator, we hypothesized that its effect could be restricted to individuals with specific INS genotypes. We found, however, no evidence for that.
The Finnish population has the highest disease incidence in the world (55.3 · 100,000eC1 · yeareC1 in 2004; A. Reunanen, personal communication), whereas the Hungarian population is among the low incidence countries in Europe (8.9 · 100,000eC1 · yeareC1) (17). In addition to this difference, the two populations also show divergence in genetic background, socioeconomic characteristics, and environment. Although the allele frequencies were somewhat different in the two populations, the disease association was consistently lacking in both series. It is unlikely that the findings in our study are explained by interpopulation differences. The statistical power of our study to detect a difference of the magnitude reported by Biason-Lauber et al. (10) at = 0.05 was more than 99%, and it was more than 80% to detect a significant OR of 1.2. To explore the striking difference between our data and those reported by Biason-Lauber et al. (10), we applied the classical 2 goodness-of-fit test for Hardy-Weinberg equilibrium (HWE) on the PAX4+1,168 genotype data from the Swiss and German population. We found that the +1,168 genotype frequencies showed severe departure from the expected values, both among the cases and control subjects (2 = 20.2, P = 0.00004; 2 = 73.9, P < 0.000001, respectively). Deviation from HWE might be seen in multiple SNPs in a strong linkage disequilibrium block, and it may also be caused by factors such as recent population migration or genetic drift. However, the most common reason for this phenomenon is genotyping error, as shown by the study of Xu et al. (18), who found that as much as 12% of the genotype data on 133 SNPs published in 75 studies were inconsistent with HWE in control subjects. Our comparison of the Swiss and German data on the +1,168 PAX4 genotype frequencies also showed that particularly the distribution of the C/A heterozygous genotype was affected by this bias, being 10.8% lower in cases and 16.2% higher among control subjects compared with the expected values. In large SNP genotyping efforts, indicators of assay quality other than HWE such as genotype call rate or Mendelian error rate may also help to reduce false-positive findings.
Holm et al. (10) observed in the 7q32 region an increase in the logarithm of odds from 2.2 to 5.3 in a pathway restricted linkage analysis using 408 Scandinavian multiplex families. They analyzed three intronic SNPs in the PAX4 gene and observed marginally significant P values in the haplotype transmission analysis. It has to be noted that these do not remain significant after correction for multiple comparisons. The T-C-C PAX4 haplotype, however, showed a P value of 0.001 in the transmission distortion test, but it was extremely rare.
In summary, we suggest that the PAX4+1,168 C/A variant does not play any essential role in genetic susceptibility to type 1 diabetes. The strong coherence between our datasets on two ethnic groups with conspicuously different disease incidences, socioeconomic characteristics, and profoundly diverse environment strengthens this finding. The striking observations made on the German and Swiss population cohorts require replication and a more detailed analysis of this gene to assess whether the difference between the two studies could be explained by heterogeneity in linkage disequilibrium patterns between ethnic groups or might reflect profound variation in the effect of PAX4 on type 1 diabetes susceptibility in particular patient groups.
ACKNOWLEDGMENTS
This study was supported by the Juvenile Diabetes Research Foundation (JDRF grants 4-1998-274 and 4-1999-731); JDRF/European Foundation for the Study of Diabetes/Novo Nordisk Focused Research Grant for Type 1 Diabetes; the Novo Nordisk Foundation; Special Federal Grants to Turku, Oulu, and Tampere University Hospitals; the Academy of Finland; and the Hungarian Scientific Research Fund (OTKA) grant T046923.
We thank Eija Nirhamo, Piia Nurmi, Mia Karlson, Ritva Suominen, and Terhi Laakso for their skillful technical assistance.
SNP, single nucleotide polymorphism
REFERENCES
Pociot F, McDermott MF: Genetics of type 1 diabetes mellitus. Genes Immun3 :235 eC249,2002
Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, MacMurray J, Meloni GF, Lucarelli P, Pellecchia M, Eisenbarth GS, Comings D, Mustelin T: A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet36 :337 eC338,2004
Guo D, Li M, Zhang Y, Yang P, Eckenrode S, Hopkins D, Zheng W, Purohit S, Podolsky RH, Muir A, Wang J, Dong Z, Brusko T, Atkinson M, Pozzilli P, Zeidler A, Raffel LJ, Jacob CO, Park Y, Serrano-Rios M, Larrad MT, Zhang Z, Garchon HJ, Bach JF, Rotter JI, She JX, Wang CY: A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet36 :837 eC841,2004
Bohren KM, Nadkarni V, Song JH, Gabbay KH, Owerbach D: A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus. J Biol Chem279 :27233 eC27238,2004
Smyth DJ, Howson JM, Lowe CE, Walker NM, Lam AC, Nutland S, Hutchings J, Tuomilehto-Wolf E, Tuomilehto J, Guja C, Ionescu-Tirgoviste C, Undlien DE, Ronningen KS, Savage D, Dunger DB, Twells RC, McArdle WL, Strachan DP, Todd JA: Assessing the validity of the association between the SUMO4 M55V variant and risk of type 1 diabetes (Comment). Nat Genet37 :110 eC111,2005
Qu H, Bharaj B, Liu XQ, Curtis JA, Newhook LA, Paterson AD, Hudson TJ, Polychronakos C: Assessing the validity of the association between the SUMO4 M55V variant and risk of type 1 diabetes. Nat Genet37 :111 eC112,2005
Dickson LM, Rhodes CJ: Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt Am J Physiol Endocrinol Metab287 :E192 eCE198,2004
Homo-Delarche F, Drexhage HA: Immune cells, pancreas development, regeneration and type 1 diabetes. Trends Immunol25 :222 eC229,2004
Servitja JM, Ferrer J: Transcriptional networks controlling pancreatic development and beta cell function (Review). Diabetologia47 :597 eC613,2004
Biason-Lauber A, Boehm B, Lang-Muritano M, Gauthier BR, Brun T, Wollheim CB, Schoenle EJ: Association of childhood type 1 diabetes mellitus with a variant of PAX4: possible link to beta cell regenerative capacity [article online]. Diabetologia48 :900 eC906,2005
Holm P, Rydlander B, Luthman H, Kockum I: Interaction and association analysis of a type 1 diabetes susceptibility locus on chromosome 5q11eCq13 and the 7q32 chromosomal region in Scandinavian families. Diabetes53 :1584 eC1591,2004
Bunce M, O’Neill CM, Barnardo MC, Krausa P, Browning MJ, Morris PJ, Welsh KI: Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens46 :355 eC367,1995
Nejentsev S, Sjoroos M, Soukka T, Knip M, Simell O, Lovgren T, Ilonen J: Population-based genetic screening for the estimation of type 1 diabetes mellitus risk in Finland: selective genotyping of markers in the HLA-DQB1, HLA-DQA1 and HLA-DRB1 loci. Diabet Med16 :985 eC992,1999
Varilo T, Paunio T, Parker A, Perola M, Meyer J, Terwilliger JD, Peltonen L: The interval of linkage disequilibrium (LD) detected with microsatellite and SNP markers in chromosomes of Finnish populations with different histories. Hum Mol Genet12 :51 eC59,2003
Walter M, Albert E, Conrad M, Keller E, Hummel M, Ferber K, Barratt BJ, Todd JA, Ziegler AG, Bonifacio E: IDDM2/insulin VNTR modifies risk conferred by IDDM1/HLA for development of type 1 diabetes and associated autoimmunity. Diabetologia46 :712 eC720,2003
Hermann R, Laine AP, Veijola R, Vahlberg T, Simell S, Lahde J, Simell O, Knip M, Ilonen J: The effect of HLA Class II, insulin and CTLA4 gene regions on the development of humoral beta cell autoimmunity. Diabetologia In press
Patterson CC, Dahlquist G, Soltesz G, Green A: Is childhood-onset type I diabetes a wealth-related disease An ecological analysis of European incidence rates. Diabetologia44 (Suppl. 3) :B9 eCB16,2001
Xu J, Turner A, Little J, Bleecker ER, Meyers DA: Positive results in association studies are associated with departure from Hardy-Weinberg equilibrium: hint for genotyping error Hum Genet111 :573 eC574,2002(Robert Hermann, Jussi Man)
2 Department of Virology, University of Turku, Turku, Finland
3 Department of Pediatrics, University of Oulu, Oulu, Finland
4 Department of Pediatrics, University of Pecs, Pecs, Hungary
5 Program of Developmental and Reproductive Biology, Biomedicum, University of Helsinki, Helsinki, Finland
6 Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland
7 Department of Pediatrics, University of Turku, Turku, Finland
8 Department of Pediatrics, Tampere University Hospital, Tampere, Finland
ABSTRACT
We aimed to assess the possible contribution of the PAX4 transcription factor gene to the genetic background of type 1 diabetes. We analyzed four coding polymorphisms of the PAX4 gene in 498 cases with type 1 diabetes and 825 control subjects from Finland and Hungary. All patients were diagnosed under the age of 15 years according to the World Health Organization criteria. All four PAX4 variants (three in exon 9 and one in exon 3) were genotyped using DNA sequencing. In addition, all Finnish subjects were typed for HLA DR-DQ, insulin gene (eC23) HphI, and CTLA4 CT60 polymorphisms. The +1,168 C/A coding variant of PAX4 was found to be polymorphic in both populations (P321H, rs712701). No difference was observed in the genotype frequencies between cases and control subjects, nor was any disease association detected when patients were stratified according to age at diagnosis, sex, HLA, insulin gene, or CTLA4 genotypes. Our data indicate that the +1,168 C/A variant of PAX4 gene does not play any essential role in genetic type 1 diabetes susceptibility. The strong coherence between the datasets of the two ethnic groups studied with highly contrasting disease incidence, socioeconomic characteristics, and profoundly diverse environment emphasizes the impact of this finding.
Type 1 diabetes is caused by immune-mediated destruction of the insulin-producing pancreatic -cells that results from a complex interplay between a polygenic background and environmental factors. In addition to the HLA complex, the insulin gene (INS), the cytotoxic T-celleCassociated protein 4 gene region (CTLA4), and the protein tyrosine phosphatase gene (PTPN22) are confirmed disease susceptibility loci (1,2). A number of other gene regions have also been found to show evidence of linkage or association with type 1 diabetes. The confirmation of true genetic effects in this complex disease remains a challenge for several reasons. One of the main hurdles is the modest disease predisposing effect of these loci (odds ratio [OR] < 2) that is further complicated by the genetic heterogeneity of the extensive patient cohorts that are required to ensure statistical power. As an example, recently two groups reported association of the small ubiquitin-like modifier 4 gene (SUMO4; OMIM 608829; 6q25) with type 1 diabetes (3,4). However, these findings were not confirmed in two large multiethnic family sets, indicating the complexity of identifying real underlying gene effects for type 1 diabetes (5,6).
The pancreatic -cell mass of an individual is tightly regulated according to insulin demand and reflects the balance between the rate of -cell replication and neogenesis and the rate of -cell apoptosis (7). During an autoimmune attack, the rate of -cell death and the capacity of -cell regeneration could be crucial factors in determining the progression of the imbalance in glucose homeostasis (8). It is known that a network of transcription factors provide genetic instructions for the differentiation and development of pancreatic -cells (9). Any alteration of this sophisticated mechanism could lead to reduced -cell mass or function and may even determine whether clinical diabetes develops.
Two recent reports have suggested that the gene encoding the pancreatic transcription factor PAX4 (OMIM 167413; 7q32) is associated with type 1 diabetes (10,11). In the current study, we aimed to assess this association in two populations with high and low incidence rates of type 1 diabetes. These populations also show conspicuous differences in terms of socioeconomic conditions, climatic, and other environmental factors.
RESEARCH DESIGN AND METHODS
All patients with type 1 diabetes were diagnosed at <15 years of age according to the World Health Organization criteria.
Finnish population series.
Children with type 1 diabetes were derived mainly from the Departments of Pediatrics at the universities in Turku, Helsinki (southern Finland), and Oulu (northern Finland) (n = 309, 50.5% males; mean age at diagnosis 8.4 ± 4.2 years). The control group comprised consecutive healthy infants born in the University Hospitals of Turku and Oulu who were randomly selected for this study (n = 535, 47.5% males).
Hungarian population series.
Patients were consecutively diagnosed in Baranya County (southern Hungary) over a 17-year period from 1980 to 1996 and recruited through the Hungarian Childhood Diabetes Registry (n = 189, 49.7% males; mean age at diagnosis 8.5 ± 4.8 years). Healthy schoolchildren born in the same geographical region and matched for sex and age were used as control subjects (n = 290, 50.7% males; mean age at sampling 10.6 ± 2.7 years).
Four single nucleotide polymorphisms (SNPs) in the PAX4 gene (OMIM 167413, 7q32) were analyzed using DNA sequencing (MegaBace1000 DNA Sequencer; GE Healthcare). Three nonsynonymous SNPs were located in exon 9 [rs712701 (P321H), rs2233583 (V330A), and rs2233584 (Ter333W)], whereas the rs2233578 SNP (R133W) was in exon 3. HLA typing was performed in all Finnish subjects using methods described earlier (12,13). The INS eC23 HphI SNP was typed with a similar oligonucleotide hybridization format (T probe 5'-Europium-CTGTCTCCCAGA-3'; A probe 5'-Terbium-CTGTCACCCAGA-3'). The CTLA4 CT60 SNP was genotyped using minisequencing (SnuPe kit; GE Healthcare). Primer sequences are available on request.
Statistical analysis.
For comparisons of genotype frequencies between the groups, 2 statistics with Yates’ correction were used. P values <0.05 were considered significant. The effect of PAX4 and the other studied genes was also examined by logistic regression analysis (SPSS for Windows, version 11.0.1; SPSS, Chicago, IL). Power calculations were done using standard methods.
RESULTS
The R133W variant in exon 3 (rs2233578) was analyzed in 46 healthy newborn infants and in 124 patients. One Finnish patient carried the arginine/tryptophan heterozygous genotype; all other subjects were arginine/arginine homozygous. At the V330A polymorphism in exon 9 (rs2233583), one child with type 1 diabetes from Finland carried the heterozygote genotype, whereas all other individuals (n = 1,322) were alanine/alanine homozygous. In addition, no variation was detected at the rs2233584 SNP (Ter333W) among the 1,323 study subjects.
The C/A variant at coding sequence position +1,168 is a nonsynonymous SNP (P321H; rs712701) that was found polymorphic. No differences in the genotype frequencies between cases and control subjects were observed in the Finnish and Hungarian series (Table 1). Accordingly, the comparison on the combined datasets showed no difference between patients and control subjects. The genotype distributions were in Hardy-Weinberg equilibrium both among cases and control subjects in the two series (P = 0.82 and 0.69, respectively, for the Finnish set; P = 0.50 and 0.96, respectively, for the Hungarian set). We stratified the Finnish patients according to age at diagnosis and sex and compared the PAX4 genotype frequencies with control subjects, but no differences were seen (Table 1). Moreover, to elucidate gene-gene interactions, we analyzed the distribution of the PAX4 genotypes in patients stratified according to the HLA DRB1-DQA1-DQB1, insulin gene (eC23) HphI, and CTLA4 CT60 genotypes and observed no differences (Table 1). The lack of association of PAX4 with type 1 diabetes was confirmed also by logistic regression analysis.
We compared the genotype and allele frequencies (rs712701 SNP) in the Finnish newborn series between northern and southern Finland, and no differences were detected (data not shown). Similarly, no regional differences were either observed in the genotype frequencies among the Finnish patients (data not shown, P = 0.69, df = 4). In contrast, the +1,168 genotype distributions showed a difference between the Finnish and Hungarian control series (P = 0.002) since the A allele was more frequent in the Finnish population (27.0 vs. 19.5% in Hungarians, P = 0.0008). As expected, genotype distributions were also different between the two populations among the patients affected by type 1 diabetes (P = 0.007).
DISCUSSION
The identification of novel type 1 diabetes genes is challenging since the disease is polygenic and the current mapping methods, such as linkage analysis, seem to have very low sensitivity, and association studies are extremely sensitive to population stratification biases. The situation is further complicated by linkage disequilibrium heterogeneity that exists between different ethnic groups (14). In addition, it is likely that there are several alternative disease pathways, and the effect of certain genetic variants is likely to be restricted to a particular molecular mechanism. Our previous studies suggest that the INS locus is associated with the emergence of insulin autoantibodies and could play a role in the development of insulin autoimmunity (15,16). In contrast, INS was not associated with multiple autoantibody positivity in children without insulin autoantibodies (16). As a consequence, new strategies for mapping of genes predisposing to type 1 diabetes are required using such cohorts, where the patients are well characterized for molecular markers such as -celleCspecific autoantibodies.
In this study, we attempted to reproduce the findings of Biason-Lauber et al. (10) on the association of the gene encoding the PAX4 transcription factor and type 1 diabetes. However, we were unable to confirm a role for the +1,168 polymorphism of PAX4 in disease pathogenesis in the two populations studied with profoundly different socioeconomic conditions, climate, and environment. Similarly, no effect of PAX4 could be seen in any patient subgroups analyzed according to age at diagnosis, sex, HLA, and CTLA4 CT60 genotypes. Since PAX4 is a pancreatic transcriptional regulator, we hypothesized that its effect could be restricted to individuals with specific INS genotypes. We found, however, no evidence for that.
The Finnish population has the highest disease incidence in the world (55.3 · 100,000eC1 · yeareC1 in 2004; A. Reunanen, personal communication), whereas the Hungarian population is among the low incidence countries in Europe (8.9 · 100,000eC1 · yeareC1) (17). In addition to this difference, the two populations also show divergence in genetic background, socioeconomic characteristics, and environment. Although the allele frequencies were somewhat different in the two populations, the disease association was consistently lacking in both series. It is unlikely that the findings in our study are explained by interpopulation differences. The statistical power of our study to detect a difference of the magnitude reported by Biason-Lauber et al. (10) at = 0.05 was more than 99%, and it was more than 80% to detect a significant OR of 1.2. To explore the striking difference between our data and those reported by Biason-Lauber et al. (10), we applied the classical 2 goodness-of-fit test for Hardy-Weinberg equilibrium (HWE) on the PAX4+1,168 genotype data from the Swiss and German population. We found that the +1,168 genotype frequencies showed severe departure from the expected values, both among the cases and control subjects (2 = 20.2, P = 0.00004; 2 = 73.9, P < 0.000001, respectively). Deviation from HWE might be seen in multiple SNPs in a strong linkage disequilibrium block, and it may also be caused by factors such as recent population migration or genetic drift. However, the most common reason for this phenomenon is genotyping error, as shown by the study of Xu et al. (18), who found that as much as 12% of the genotype data on 133 SNPs published in 75 studies were inconsistent with HWE in control subjects. Our comparison of the Swiss and German data on the +1,168 PAX4 genotype frequencies also showed that particularly the distribution of the C/A heterozygous genotype was affected by this bias, being 10.8% lower in cases and 16.2% higher among control subjects compared with the expected values. In large SNP genotyping efforts, indicators of assay quality other than HWE such as genotype call rate or Mendelian error rate may also help to reduce false-positive findings.
Holm et al. (10) observed in the 7q32 region an increase in the logarithm of odds from 2.2 to 5.3 in a pathway restricted linkage analysis using 408 Scandinavian multiplex families. They analyzed three intronic SNPs in the PAX4 gene and observed marginally significant P values in the haplotype transmission analysis. It has to be noted that these do not remain significant after correction for multiple comparisons. The T-C-C PAX4 haplotype, however, showed a P value of 0.001 in the transmission distortion test, but it was extremely rare.
In summary, we suggest that the PAX4+1,168 C/A variant does not play any essential role in genetic susceptibility to type 1 diabetes. The strong coherence between our datasets on two ethnic groups with conspicuously different disease incidences, socioeconomic characteristics, and profoundly diverse environment strengthens this finding. The striking observations made on the German and Swiss population cohorts require replication and a more detailed analysis of this gene to assess whether the difference between the two studies could be explained by heterogeneity in linkage disequilibrium patterns between ethnic groups or might reflect profound variation in the effect of PAX4 on type 1 diabetes susceptibility in particular patient groups.
ACKNOWLEDGMENTS
This study was supported by the Juvenile Diabetes Research Foundation (JDRF grants 4-1998-274 and 4-1999-731); JDRF/European Foundation for the Study of Diabetes/Novo Nordisk Focused Research Grant for Type 1 Diabetes; the Novo Nordisk Foundation; Special Federal Grants to Turku, Oulu, and Tampere University Hospitals; the Academy of Finland; and the Hungarian Scientific Research Fund (OTKA) grant T046923.
We thank Eija Nirhamo, Piia Nurmi, Mia Karlson, Ritva Suominen, and Terhi Laakso for their skillful technical assistance.
SNP, single nucleotide polymorphism
REFERENCES
Pociot F, McDermott MF: Genetics of type 1 diabetes mellitus. Genes Immun3 :235 eC249,2002
Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, MacMurray J, Meloni GF, Lucarelli P, Pellecchia M, Eisenbarth GS, Comings D, Mustelin T: A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet36 :337 eC338,2004
Guo D, Li M, Zhang Y, Yang P, Eckenrode S, Hopkins D, Zheng W, Purohit S, Podolsky RH, Muir A, Wang J, Dong Z, Brusko T, Atkinson M, Pozzilli P, Zeidler A, Raffel LJ, Jacob CO, Park Y, Serrano-Rios M, Larrad MT, Zhang Z, Garchon HJ, Bach JF, Rotter JI, She JX, Wang CY: A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet36 :837 eC841,2004
Bohren KM, Nadkarni V, Song JH, Gabbay KH, Owerbach D: A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus. J Biol Chem279 :27233 eC27238,2004
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