type 1 diabetes mellitus ( dm ) and graves disease are autoimmune diseases , and a number of 
 genetic and environmental factors are thought to be involved in their etiology .
we have 
 demonstrated an association between both hla class ii genotype and ctla-4 
 ( cytotoxic t lymphocyte antigen-4 ) gene polymorphism and childhood - onset type 1 dm ( 1 , 2 ) . the 
 incidences of hla - drb1 * 0405 , * 0901 , and dqb1 * 0303 , * 0401 were found to be significantly 
 higher in children with type 1 dm than in controls , whereas the incidences of drb1 * 0803 , 
 * 1501 , * 1502 and dqb1 * 0301 , * 0601 , * 0602 were significantly lower ( 1 ) .
the g allele in the ctla-4 a / g polymorphism at 
 position 49 was more frequent in type1 dm children than in controls ( 2 ) .
we have also found an association between both hla class ii genotype 
 and ctla-4 gene polymorphism and childhood - onset graves disease in 
 japanese patients ( submitted for publication ) . in 1994
aaltonen et al . assigned the disease locus in finnish families 
 with apeced ( autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy ) to chromosome 
 21q22.3 based on the results of a linkage analysis between two markers , d21s49 and d21s171 
 ( 3 ) , and in 1997 a novel gene was isolated from this 
 region and named the aire-1 ( autoimmune regulator-1 ) gene ( 4 , 5 ) . the 
 aire-1 gene is composed of 14 exons .
aire-1 protein is expressed in the 
 thymic medulla , where t - cell immune tolerance is established , as well as in the lymph nodes , 
 spleen , and fetal liver , but it is not expressed in the affected organs of apeced patients . 

kogawa et al . recently found that aire-1 protein is also restrictively 
 expressed in peripheral cd14-positive monocytes and in differentiated dendritic cells ( 6 ) .
apeced is an autosomal recessive disease that is especially frequent in finnish and iranian 
 jews , and is characterized by the simultaneous presence of at least two of three major 
 diseases in the same individual : hypoparathyroidism , addison s disease , and chronic 
 mucocutaneous candidiasis .
it may also be associated with clinical manifestations of 
 autoimmune thyroid disease , type 1 dm , and gonadal dysfunction .
apeced causes multiple organ 
 dysfunction in a wide variety of endocrine and extra - endocrine organs , and production of 
 autoantibodies against the affected organs and lymphocyte invasion of the affected organs 
 have frequently been demonstrated .
a high percentage of patients with type 1 dm produce autoantibody against the thyroid gland 
 as well as against the  cells of the pancreas and have autoimmune thyroid 
 disease , including graves disease and hashimoto thyroiditis ( 7 , 8) .
the patients with graves disease may 
 not only have autoantibodies against thyroid epithelial cells or the tsh receptor , but 
 antinuclear antibodies and antineutrophil cytoplasmic antibodies ( anca ) that are not 
 specific for endocrine organs ( 9 ) .
these findings suggest that patients with type 1 dm and graves disease tend to have 
 autoimmune reactions not only against specific target organs but against several other 
 organs and that they may possess heterozygous aire-1 mutations . in this 
 study we examined japanese children with isolated type 1 dm or graves disease for the 
 presence of heterozygous aire-1 mutations . as a first step we analyzed the 
 r257x heterozygous mutation in exon 6 and the k83e heterozygous mutation in exon 2 which can 
 be easily analyzed .
the r257x mutation is the most common mutation in apeced which has been 
 reported in many ethnic groups and in exon 2 many kinds of missense mutation have been 
 reported .
forty - six unrelated children with type 1 dm ( 17 males and 29 females ; age at the time of 
 diagnosis , 0.516 yr ) and 44 unrelated children with graves disease ( 10 males and 34 
 females ; age at the time of diagnosis , 316 yr ) were the subjects of this study .
type 1 dm was diagnosed according to the criteria of the who study group ( 10 ) .
the age of the subjects with type 1 dm at the time 
 of diagnosis ranged from six mo to 16 yr .
the interval between the time of diagnosis and 
 examination for mutations ranged widely , from 0 to 18 yr .
twenty - one of the 39 dm patients 
 tested were positive for anti - gad antibody , and 20 of the 34 dm patients tested were 
 positive for ia-2 antibody .
graves disease was diagnosed on the basis of the presence of biochemical hyperthyroidism , 
 the presence of tsh receptor antibodies ( trab ) , and the presence of clinical evidence , such 
 as palpable diffuse goiter , exophthalmos , or tachycardia , plus the absence of other causes 
 of hyperthyroidism .
the age of the graves disease patients at the time of diagnosis ranged 
 from 3 to 16 yr .
the microsome 
 test ( mchc ) was positive in 37 of the 44 graves disease patients , and the thyroid test 
 ( tgha ) was positive in 10 of the 44 .
this study was carried out in accordance with the principles of the declaration of 
 helsinki .
in this study we analyzed aire-1 genes for an a > g transition in exon 2 
 ( k83e mutation ) and a c > t transition in exon 6 ( r257x mutation ) .
dna was extracted from 
 peripheral blood leukocytes with dna quick ii ( dainihonseiyaku , japan ) .
the 
 aire-1 gene mutations were typed by pcr of genomic dna and restriction 
 fragment - length polymorphism analysis ( pcr - rflp ) ( 4 ) .
the primers used for the k83e mutation were 5-tccaccacaagccgaggagat-3 and 
 5-acgggctcctcaaacaccact-3. the pcr was performed in a thermocycler at 94c for 5 min 
 followed by 35 cycles of 94c for 30 sec , 60c for 30 sec and 68c for 30 sec , and a final 
 step at 68c for 10 min .
the amplified products were dissolved with restriction enzyme 
 taqi ( roche , germany ) and run on a 2% agarose gel .
the a allele 
 corresponds to the 424-base pair ( bp ) uncleaved fragment with no taqi site 
 and the g allele corresponds to the presence of 261-bp and 163-bp cleaved fragments 
 generated by taqi dissolution .
the primers used for the r257x mutation were 5-gcggctccaagaagtgcatccagg-3 and 
 5-ctccaccctgcaaggaagaggggc-3. the pcr was performed in a thermocycler at 94c for 5 min 
 followed by 35 cycles of 94c for 30 sec , 65c for 30 sec and 72c for 30 sec , and a final 
 step at 72c for 10 min .
the amplified products were dissolved with restriction enzyme 
 taqi and run on polyacrylamide gel .
the c allele corresponds to the 
 presence of three cleaved fragments ( 222 bp , 61 bp , and 55 bp ) , and the t allele corresponds 
 to the presence of two cleaved fragments ( 283 bp and 55 bp ) generated by 
 taqi dissolution ( 4 ) .
1 electrophetograms showing the results of digestion with taq1 . ( a ) analysis for the 
 a > g transition ( k83e mutation ) in exon 2 . figure ( a ) shows the 424-bp uncleaved 
 fragment in two representative samples of type 1 dm and graves disease ( gd ) subjects . 

( b ) analysis for the c > t transition ( r256x mutation ) in exon 6 . figure ( b ) shows 
 three cleaved fragments ( 222 bp , 61 bp , and 55 bp ) in two representative samples of 
 type 1 dm and graves disease ( gd ) subjects .. analysis of the a > g transition in exon 2 ( k83e mutation ) revealed the 424-bp 
 uncleaved fragment in all of the type 1 dm and graves disease subjects , and analysis of the 
 c > t transition in exon 6 ( r257x mutation ) revealed three cleaved fragments ( 222 bp , 61 
 bp , and 55 bp ) in all of the subjects . none of the childhood - onset type 1 dm or graves 
 disease subjects had the k83e or r257x heterozygous mutation in the aire-1 
 gene .  
( a ) analysis for the 
 a > g transition ( k83e mutation ) in exon 2 .
figure ( a ) shows the 424-bp uncleaved 
 fragment in two representative samples of type 1 dm and graves disease ( gd ) subjects . 

( b ) analysis for the c > t transition ( r256x mutation ) in exon 6 . figure ( b ) shows 
 three cleaved fragments ( 222 bp , 61 bp , and 55 bp ) in two representative samples of 
 type 1 dm and graves disease ( gd ) subjects .
meyer et al . examined 139 patients with graves disease , 224 with type 1 
 dm , 83 with addison s disease , and 75 with hashimoto s thyroiditis , in germany , for an 
 association between heterozygous aire-1 mutations and isolated autoimmune 
 endocrinopathy ( 11 ) .
the r257x mutation was found in 
 one patient with hashimoto s thyroiditis in its heterozygous form , but not in any patients 
 with type 1 dm , graves disease , or addison s disease .
it was noteworthy that the patient 
 who had the r257x mutation in its heterozygous form later developed other endocrine 
 diseases . in the present study in japanese patients
we did not find any k83e or r257x heterozygous 
 mutations of the aire-1 gene in 46 children with type 1 dm and 44 children 
 with graves disease .
these results suggest the absence of any association between k83e or 
 r257x heterozygous mutations and isolated type 1 dm and graves disease in japanese 
 children .
although apeced is very rare in japan , the following mutations have been discovered in 
 japanese patients : l28p in exon 1 and ivs9 - 1g > c ( 12 ) , missense mutation r15c in exon 1 ( 13 ) , 
 insertion of a cytosine at nucleotide 29635 in exon 10 , and a deletion of guanine at 
 nucleotide 33031 in exon 13 ( 14 ) .
however , there have 
 been no reports of japanese apeced patients with r257x or k83e mutations .
an analysis of the 
 heterozygous mutations that have been discovered in japanese apeced patients should be 
 included in future studies of genetic factors in childhood - onset type 1 dm and graves 
 disease in japanese patients . in conclusion ,
no k83e and r257x heterozygous mutations in the aire-1 gene 
 were found in japanese children with isolated type 1 dm or graves disease .
further study is 
 needed to conclude whether there is an association between heterozygous 
 aire-1 gene mutations or polymorphisms and isolated type 1 dm or graves 
 disease in japanese patients .
 type 1 diabetes mellitus ( dm ) and graves disease are autoimmune diseases , and 
 a number of genetic factors , including hla and ctla-4 genes , have been reported to 
 contribute to their etiology . 
 the gene responsible for autoimmune polyendocrinopathy- 
 candidiasis - ectodermal dystrophy ( apeced ) has been cloned and named the autoimmune 
 regulator-1 ( aire-1 ) gene . aire-1 protein is thought to be a 
 transcription regulatory protein and to have a role in the maintenance of immunological 
 tolerance . 
 the aim of this study was to determine whether heterozygous 
 aire-1 gene mutations are associated with childhood - onset type 1 
 diabetes and graves disease in the japanese population . 
 we investigated 46 children with 
 type 1 dm ( 29 females and 17 males ; age at the time of diagnosis , 0.516 yr ) and 44 
 children with graves disease ( 34 females and 10 males ; age at the time of diagnosis , 316 
 yr ) for the presence of the k83e mutation in exon 2 and the r257x mutation in exon 6 of 
 the aire-1 gene . 
 the alleles were identified by polymerase chain reaction 
 of genomic dna and restriction fragment - length polymorphism analysis ( pcr - rflp ) with 
 endonuclease taqi . 
 since no patients with type 1 dm or graves disease 
 were found to carry the k83e or the r257x heterozygous mutation , we concluded that neither 
 the k83e nor the r257x heterozygous mutation in the aire-1 gene seem to 
 be the cause of the more common isolated endocrinopathies , i.e. , type 1 diabetes mellitus 
 and graves disease , in japanese children . 
Introduction
Subjects
Method
Results
Discussion