the prevalence of 
 newborns with hyperthyrotropinemia was 1 in 8,000 live births at the beginning of neonatal screening in 1979 
 ( 1 ) , and it is now 1 in 2,500 births ( 2 ) in japan . due to the increase in the prevalence of screening - positive newborns ,
the number of 
 newborns diagnosed as having transient hypothyroidism or transient hyperthyrotropinemia has increased .
it has 
 been reported that about 30% of screening - positive newborns with anatomically normal thyroids have transient 
 forms of the disorder ( 3 ) .
the causes of transient hypothyroidism or 
 transient hyperthyrotropinemia include prematurity , maternal thyroid disease and excess or lack of maternal 
 iodine intake ( 4 ) . on the other hand
, it has been reported that 
 subclinical hypothyroidism persists in late childhood in about 30% of children found to be false - positive during 
 neonatal screening ( 5 ) .
it is unclear whether transient thyroid dysfunction and subclinical hypothyroidism represent true clinical 
 conditions .
the aim of this study was to determine whether transient thyroid dysfunction and subclinical 
 hypothyroidism detected during neonatal screening are influenced by genetic background .
the tsh receptor ( tshr ) , 
 thyroid peroxidase ( tpo ) and dual oxidase 2 ( duox2 ) genes , for which it has been reported heterozygous defects 
 cause neonatal transient thyroid dysfunction , were analyzed in children with transient thyroid dysfunction or 
 subclinical hypothyroidism detected during neonatal screening .
we recruited nine screening - positive children who had not received levothyroxine ( l - t4 ) replacement or had been 
 able to stop l - t4 replacement after re - evaluation of thyroid function .
subjects with known underlying causes of 
 transient thyroid dysfunction , such as prematurity , down syndrome , maternal thyroid disease and excessive 
 maternal iodine intake , were excluded .
they were continuously observed at toho university omori medical center 
 or ibaraki children s hospital .
the study was performed with the approval of the institutional review boards of the toho university school of 
 medicine and ibaraki children s hospital , and informed consent for participation in this study was obtained from 
 all the subjects parents .
all exons of the tshr 
 gene , tpo gene and duox2 gene were amplified by polymerase chain reaction ( pcr ) using primers described in 
 previous reports ( 6,7,8,9 ) .
the purified pcr products were 
 sequenced directly by an automated dna sequence analyzer ( abi 310 autosequencer , applied biosystems , foster 
 city , ca , usa ) .
basal tsh levels above 5 u / ml were considered elevated ( 5 ) , and peak tsh 
 levels after trh stimulation above 35 u / ml in infants and above 2530 u / ml in children were considered 
 exaggerated responses ( 10 , 11 ) .
the serum tsh level was 24.3  11.2 u / ml ( n=8 ) during neonatal screening .
the serum tsh and free t4 levels 
 during the first visit to the hospital at 27  16 d of life were 14.3  8.0 u / ml ( n=9 ) and 1.52  0.25 ng / dl 
 ( n=8 ) , respectively ( table 1 ) .
four children had not 
 received l - t4 replacement , but their thyroid functions had been evaluated every 6 to 12 mo .
although subject 8 
 showed normal serum tsh and t4 levels ( 4.19 u / ml and 10.9 g / dl , respectively ) during her first visit to the 
 hospital at 56 d of life , her serum thyroglobulin ( tg ) level ( 120 ng / ml ) was slightly high .
the remaining five children started l - t4 replacement during the neonatal 
 period and stopped it after re - evaluation of thyroid function at the age of 5.5  2.2 yr .
after the cessation of 
 l - t4 replacement , their thyroid functions were evaluated every 6 mo .
the initial dose of l - t4 replacement was 
 3.2  0.5 g / kg / d , and the dose of l - t4 replacement at cessation was 1.0  0.2 g / kg / d ( table 2 ) .
no 
 patients had a family history of thyroid diseases.table 1   subjects initial thyroid function resultssubjectsexserum tsh level at screening ( u / ml)at first visit to the hospitalinitial dose of levothyroxine ( l - t4 ) 
 ( g / kg / d)age ( d)basal tsh level ( u / ml)ft4 ( ng / ml)tsh peak after trh stimulation ( u / ml)1f42.01217.961.7190.713.42f15.41711.881.5948.762.63f14.5545.51.2nano treatment4f26.11832.781.75na3.05m14.51214.321.0952.913.86m43.41119.051.8546.623.07m20.33811.581.42nano treatment8fna564.19nanano treatment9m17.92011.81.2nano treatmentna : not available . 
 table 2   subjects follow - up thyroid function resultssubjectagegenetic defectbasal tsh level under no treatment 
 ( u / ml)at re - evaluationagetsh peak after trh ( u / ml)dose of l - t4 ( g / kg / d)16yr , 5moa single tshr mutation ( r450h)3.206.004yr , 0mo43.71.028yr , 4mond1.402.806yr , 6mo21.70.938yr , 4mond2.98nano treatment48yr , 6mond2.502.607yr , 2mo18.41.359yr , 3mond2.106.605yr , 6mo29.10.8610yr , 4mond1.401.909yr , 0mo20.71.0714yr , 1mond1.206.550yr , 8mo26.9no treatment814yr , 3moa single tpo mutation ( p883s)3.016.1110yr , 8mo30.2no treatment915yr , 0mond1.305.001yr , 5mo22.5no treatmentnd : not detected , na : not available . nd : not detected , na : not available .
ultimately , subjects 1 , 5 , 7 and 8 were diagnosed as having subclinical hypothyroidism because of fluctuating 
 basal tsh levels during the no treatment period and/or exaggerated tsh responses to trh simulation at 
 re - evaluation . all subjects with subclinical hypothyroidism were negative for anti - tpo and anti - tg antibodies . 

subjects 2 , 4 and 6 were diagnosed as having neonatal transient hypothyroidism because they showed normal tsh 
 responses to trh stimulation at re - evaluation and their basal tsh levels had been normal after cessation of l - t4 
 replacement .
subjects 3 and 9 were diagnosed as having transient hyperthyrotropinemia because they had not 
 received l - t4 replacement and their basal tsh levels had been normal since the early infantile period .
subject 1 was heterozygous for a tshr gene mutation ( r450h ) , and subject 8 was heterozygous for a tpo gene 
 mutation ( p883s ; fig .
1 sequence analysis of a tshr gene in subject 1 
 ( a ) and a tpo gene in subject 8 ( b ) . (
a ) the mutant site ( g to a ) is indicated by the arrow .
( b ) the mutant site ( c to t ) is indicated by the arrow . 

sequence analysis of a tshr gene in subject 1 
 ( a ) and a tpo gene in subject 8 ( b ) .
( a ) the mutant site ( g to a ) is indicated by the arrow .
( b ) the mutant site ( c to t ) is indicated by the arrow . 

one group 
 consists of genes causing thyroid dysgenesis , and the other group consists of genes causing thyroid 
 dyshormonogenesis ( 12 ) . among them , tshr , sodium iodine symporter ( nis ) , 
 tg , tpo and duox2 gene defects have been occasionally reported to cause mild hypothyroidism .
subjects who are 
 heterozygous for tshr gene mutations have been reported to show various phenotypes , from euthyroid to mild 
 hypothyroidism ( 13,14,15,16 ) .
some subjects with 
 heterozygous tshr gene mutations were identified during neonatal screening , and the others were identified in 
 the course of evaluation for common nonspecific symptoms .
subjects with biallelic inactivating mutations of the 
 nis gene and tg gene have also been reported to show various phenotypes , from euthyroid to severe hypothyroidism 
 ( 17,18,19,20 ) .
reported that the 
 heterozygous carrier rate for tpo gene mutations is significantly higher in babies with neonatal transient 
 hypothyroidism than in normal babies and suggested that the presence of heterozygous tpo gene mutations 
 contributes to development of neonatal transient hypothyroidism ( 21 ) . 

several reports suggest that biallelic inactivating mutations in the duox2 gene are associated with severe and 
 permanent congenital hypothyroidism and that heterozygous mutations of duox2 gene are associated with neonatal 
 transient hypothyroidism ( 9 , 22 ) . 

however , maruo et al . reported that subjects with compound heterozygous duox2 mutations 
 detected during neonatal screening also showed transient hypothyroidism ( 23 ) . in the present study , the tshr , tpo and duox2 genes were analyzed because monoallelic mutations of these genes 
 might cause transient neonatal thyroid dysfunction .
two of nine children with transient thyroid dysfunction or 
 subclinical hypothyroidism detected during neonatal screening had heterozygous gene mutations , one for a tshr 
 gene ( r450h ) and one for a tpo gene ( p883s ) .
the r450h mutation of the tshr gene is common in japan ( 15 ) , and the p883s mutation of the tpo gene has been reported in japanese 
 patients ( 24 ) . since nis and tg genes were not analyzed in the present 
 study , some of the remaining seven children may have had mutations of these two genes .
although further 
 examinations are necessary , genetic background may contribute to development of the transient thyroid 
 dysfunction and subclinical hypothyroidism detected during neonatal screening .
thyroid function is influenced by several factors , such as general condition , age and amount of iodine intake . 

it is well known that patients with subclinical hypothyroidism can develop overt hypothyroidism during puberty 
 and pregnancy due to the increase in thyroid hormone requirement .
however , large amounts of iodine intake have been reported to cover the development of 
 hypothyroidism in patients with biallelic inactivating mutations of the nis and duox2 genes ( 17 , 25 ) .
moreover , it has been 
 reported that , if left untreated , goiters caused by elevated tsh stimulation in patients with defects of the tpo 
 gene and tg gene are prone to development into thyroid cancer ( 26 , 27 ) .
therefore , it is important to continue long - term follow - up of children 
 with transient thyroid dysfunction and subclinical hypothyroidism detected during neonatal screening to avoid 
 missing the development of overt hypothyroidism and goiters , given that they may have background gene 
 defects . in conclusion ,
two of the nine screening - positive children who had not received l - t4 replacement or had been 
 able to stop the treatment had genetic mutations .
it is possible that genetic background contributes to the 
 development of transient thyroid dysfunction and subclinical hypothyroidism detected during neonatal screening . 

therefore , long - term follow - up of screening - positive children is required .
 about 30% of children with elevated tsh levels during neonatal screening have a transient form of disorder . 
 on the other hand , it has been reported that subclinical hypothyroidism persists in late childhood in about 
 30% of children found to be false - positive during neonatal screening . 
 the aim of this study was to determine 
 whether transient thyroid dysfunction and subclinical hypothyroidism detected during neonatal screening are 
 influenced by genetic background . 
 the tsh receptor ( tshr ) , thyroid peroxidase ( tpo ) and dual oxidase 2 ( duox2 ) 
 genes , for which it has been reported that heterozygous defects cause neonatal transient thyroid dysfunction , 
 were analyzed . 
 nine children with transient thyroid dysfunction or subclinical hypothyroidism detected during 
 neonatal screening were studied . 
 one child was heterozygous for a tshr gene mutation ( r450h ) , and another 
 child was heterozygous for a tpo gene mutation ( p883s ) . 
 no children with mutation of the duox2 gene were 
 identified . 
 genetic background may contribute to development of transient thyroid dysfunction and subclinical 
 hypothyroidism detected during neonatal screening . 