1 Dipartimento di Scienze Biomediche e Biotecnologie, Università di Cagliari, Ospedale Microcitemico, Cagliari, Italy
2 Servizio di Diabetologia Pediatrica, Ospedale G. Brotzu, Cagliari, Italy
3 Istituto di Clinica Medica, Servizio di Diabetologia, Universita di Sassari, Sassari, Italy
4 Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Addenbrookes Hospital, Cambridge, U.K
5 Centro di Genetica Clinica, Dipartimento di Scienze Biomediche, Universita di Sassari, Sassari, Italy
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ABSTRACT |
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It is believed that most cases of type 1 diabetes result from an autoimmune, T-celldependent destruction of the insulin-producing pancreatic ß-cells and subsequent irreversible insulin deficiency. Autoimmune diabetes is more commonly inherited as a common multifactorial trait but can also occur in two rare monogenic disorders, APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) and IPEX, both of which are characterized by a severe autoimmune pathology of several organs and tissues. The FOXP3 gene and the mouse orthologue Foxp3 are members of a gene family that encode transcription factors possessing a winged helix or forkhead box ("fox") DNA-binding domain. It has been recently shown that Foxp3 represents a key regulator of the development and function of a subset of CD4 regulatory T-cells, which express the interleukin-2 receptor CD25, and are central in the regulation of both the adaptive and innate immune system (14). The elucidation of the molecular bases of these rare Mendelian disorders has provided insights into the etiology of autoimmunity in humans and in mice (2,3,59). It is possible that common DNA polymorphisms of FOXP3 also influence susceptibility to the common, multifactorial form of type 1 diabetes. This hypothesis was strengthened by the observation that in common type 1 diabetes in Sardinia there is a strong male bias in disease incidence, and evidence of linkage of disease to the same region of chromosome X that encodes FOXP3 (10,11) has been observed. Furthermore, we have excluded the involvement of a Y-chromosome gene as being the cause of the observed male excess of type 1 diabetes in Sardinian patients (11).
Taken together, these observations suggest that if common variants exist that change the function or expression of FOXP3 in more subtle ways than the described rare, highly penetrant mutations, these could help explain the elevated male-to-female ratio in young-onset cases of common type 1 diabetes and its potential linkage to chromosome X. The aim of this study was, therefore, to test if common variation at FOXP3 was associated with the common form of type 1 diabetes in Sardinia. We initially characterized the FOXP3 region content of single nucleotide polymorphisms (SNPs) and other polymorphisms by sequencing the gene in a panel of 64 male Sardinian individuals and by supplementing and enlarging the map using polymorphisms from public databases (see RESEARCH DESIGN AND METHODS). In particular, we sequenced an overall 5.7 kb per individual, including all of the 11 coding exons and surrounding intron-exon boundaries as well as the fragments upstream of the first coding exon and downstream of exon 11, respectively, revealing no exonic or obvious splice variants. Twelve polymorphisms, including seven SNPs (one novel detected by resequencing the gene), two deletions, and three additional polymorphic markers, spanning an 82-kb FOXP3 region were selected and genotyped in 418 Sardinian type 1 diabetic families (RESEARCH DESIGN AND METHODS and Fig. 1). In this family dataset we have, therefore, tested each marker for disease association by analyzing the maternal meioses with the transmission disequilibrium test and the paternal meioses using a test that we refer to as AFBAXPAT (affected familybased X-chromosome paternal association test) (see RESEARCH DESIGN AND METHODS). These analyses did not show any evidence of disease association at the 5% level of significance (data not shown, presented in the online appendix [available from http://diabetes.diabetesjournals.org). The intermarker linkage disequilibrium patterns indicate that there is no evidence of a break in the linkage disequilibrium between contiguous markers and, therefore, that it is unlikely that we have failed to detect a disease association due to any polymorphism in this region that was not genotyped (Fig. 2).
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We conclude that variation at, or near, FOXP3 is not associated with type 1 diabetes in Sardinia and on its own cannot explain the male-female bias in disease incidence in this population. These results strongly support the view that variation at, or close to, FOXP3 exons does not play a major role in the familial clustering and in general population risk of the common multifactorial form of type 1 diabetes. However, we cannot exclude that regulatory variation distant from FOXP3 and not in linkage disequilibrium with any of the variants tested here might affect the expression of this gene and influence the disease risk.
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RESEARCH DESIGN AND METHODS |
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A subset of 375 type 1 diabetic patients was assayed for the presence of antithyroid peroxidase antibodies indicative of an autoimmune reaction against the thyroid gland. They were also assayed for the presence of anti-endomysial and anti-transglutaminase antibodies as a screening assessment for the presence of celiac disease. Patients who had antithyroid peroxidase antibody values >100 units/ml were considered to be positive in this study. Patients found positive for the presence of anti-endomysial and/or anti-transglutaminase antibodies were subjected to intestinal biopsy and to a standard diagnostic gluten-free/gluten trial before establishing a diagnosis of celiac disease.
FOXP3 polymorphism content.
We characterized the FOXP3 region content of SNPs and other polymorphisms, such as microsatellites and microdeletions. We resequenced the gene in a panel of 64 male individuals, 32 type 1 diabetic patients and 32 healthy control subjects: all 11 coding exons and surrounding intron-exon boundaries, as well as the regions 1,330 bp upstream of the first exon and 988 bp downstream of exon 11, comprising a total of 7.5 kb per individual DNA. To sequence these regions, we designed 13 pairs of primers for PCR fragments (reported in the online appendix). We identified two novel variants that are referred to as, respectively, N1: C>T, 172 bp 3' from exon 11, and N2: G>A, 673 bp 3' from exon 11. One of these novel variants, N2, was extremely rare, being present in only one control subject, and was not further typed. The N1 variant, being present in four patients and two control subjects, was instead more common and was therefore typed and tested for association with type 1 diabetes together with other 11 known polymorphisms (see below). The position and sequence of these 11 polymorphisms, which included six SNPs, three microsatellite markers, and two insertions/deletions, were obtained from public databases (accession number AF235097, http://www.ncbi.nlm.nih.gov).
Genotyping.
The DNA fragments containing the six known SNPs were amplified by PCR, and the products that were dot blot analyzed using primers and the sequence-specific oligonucleotide probes are reported in the online appendix. The novel SNP was genotyped by using the MGB TaqMan technology (ordered to the Applera by the "assay by design"). The other five polymorphisms (microsatellites and deletions) were genotyped by separating fluorescently tagged PCR products on a 96-capillary sequencer (MegaBACE 1000) using the Genetic Profiler software (Amersham-Pharmacia Biotech, Buckinghamshire, U.K.). The primers used to amplify these fragments are also reported in the online appendix. Numerical values (1, 2, 3, etc.) were given to microsatellite alleles starting from the allele with the lowest number of repeats.
Statistical analysis.
The 12 polymorphisms of interest were typed and tested for disease association in the dataset of 418 type 1 diabetic families. To this aim, we adapted the transmission disequilibrium test to an X-linked inheritance by evaluating any departure from a random expectation of 50% in the transmission of alleles or haplotypes from heterozygous mothers to affected children (13). Because the male excess observed in type 1 diabetic Sardinian patients (10,11) is consistent with an X-linked recessive inheritance, we reasoned that paternal contribution to affected daughters could also be important for the full expression of the disease risk. In order to evaluate the paternal meioses, we developed a new test, which we refer to as AFBAXPAT. In this new test, the "disease chromosomes" are represented by the variants (alleles and haplotypes) obligatorily transmitted from the fathers to the affected daughters, whereas the "control chromosomes" are represented by the variants of the fathers having only affected male children (i.e., with no affected daughters). The disease and control alleles and haplotypes were then arranged in a 2 x 2 contingency table and compared using a 2 test under the null hypothesis of no marker association with the disease and assuming random mating and Hardy-Weinberg equilibrium.
The five most informative polymorphisms, based on evaluation of the linkage disequilibrium between variants, were also analyzed with a case-control design using a larger dataset that included both the 418 families and the case-control dataset of 268 male patients and 326 healthy male individuals. In this mixed dataset, the "disease chromosomes" are represented by the variants inherited by the affected children (only probands in families with more than one affected sibling were considered), whereas the control "population" is constructed from three sources: 1) the variants detected in the 326 male blood donors, 2) the variants not transmitted from the mothers to the affected child (or in the case of multiplex families, never transmitted to any affected child), and 3) the variants detected in the fathers having only affected male children as outlined in the above-described AFBAXPAT. The disease and control allele and haplotype counts were compared using a 2 test under the null hypothesis of no marker association with the disease and assuming random mating and Hardy-Weinberg equilibrium. The statistical power of our sample set has been computed, considering the individual frequencies of the various alleles in the general population, based on standard epidemiological measures applied to 2 x 2 contingency tables.
The choice of the markers to be followed up in the case-control dataset was reached empirically by sorting the observed haplotypes and selecting the most informative variants and removing the redundant ones. The selection and definition of the relative redundancy of each marker were assisted using the intermarker linkage disequilibrium estimates and the software CZAllClust, written by C. Zavattari and available at http://mcweb.unica.it/immunogeneticslab. The linkage disequilibrium patterns between the marker loci assessed in this study were calculated on the parental chromosomes from 418 families, using a normalized disequilibrium (total D'), multiallelic extension of Lewontins standardized measure of disequilibrium (14,15). The D' values range from 0 to 1, with 0 reflecting perfect independence between alleles at the two loci compared and 1 reflecting complete linkage disequilibrium (14,16). The multiallelic D' value was calculated by the program haploxt, which is available at http://archimedes.well.ox.ac.uk/pise/haploxt-simple.html. The respective P values were calculated using the Markov chain method described by Guo and Thompson (15) (available at http://anthropologie.unige.ch/arlequin). In all cases, 100,000 tables were explored. Unequivocal, phase-known haplotypes were established by following the cosegregation of alleles within the type 1 diabetic families or by selecting male individuals.
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ACKNOWLEDGMENTS |
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We thank Antonio Cao for continuous support and encouragement. We also thank Bob Wildin for sharing information early in the study; Jamie Foster, Jennifer Masters, and Jason Cooper for help and advice; Margi Chessa, Rossella Ricciardi, and Adolfo Pacifico for help in collecting the Sardinian type 1 diabetic families and for clinical information; Cesare Zavattari for writing CZAllClust; and Maria Melis and Antonella Deidda for taking the blood of the patients and their relatives.
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FOOTNOTES |
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Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
Address correspondence and reprint requests to Francesco Cucca, Dipartimento di Scienze Biomediche e Biotecnologie, University of Cagliari, Via Jenner, Cagliari 09121, Italy. E-mail: fcucca{at}mcweb.unica.it
Received for publication March 19, 2004 and accepted in revised form April 19, 2004
AFBAXPAT, affected familybased X-chromosome paternal association test; FOX, forkhead/winged helix transcription factor; SNP, single nucleotide polymorphism; TPOA, autoantibody to thyroid peroxidase
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REFERENCES |
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