neonatal diabetes mellitus ( ndm ) occurs within the first six months of life . depending on clinical outcomes , it is classified into
transient neonatal diabetes mellitus ( tndm ) and permanent neonatal diabetes mellitus ( pndm ) .
tndm , which accounts for 50% to 60% of ndm , goes into remission after treatment for an average period of 12 weeks .
tndm is usually diagnosed within one month after birth with a median age at diagnosis of 6 days .
it is characterized by intrauterine growth retardation ( iugr ) , less frequent diabetic ketoacidosis , requirement of low initial dose of insulin for treatment , and early remission . about 50% of tndm patients , however , may have relapse in adulthood and require lifelong insulin maintenance therapy
. the clinical features of tndm and pndm overlap , and the typing is based on clinical remission on follow - up .
more than 20 pathogenic genes have been identified in pndm , of which the most common are kcnj11 and abcc8 encoding the kir6.2 and sur1 subunits of katp channel accounting for 40% to 60% [ 4 , 5 ] .
mutations in kcnj11 and abcc8 lead to persistence of katp channel in the open state inducing membrane hyperpolarization and impaired insulin secretion .
other infrequent mutations include gck , pdx1 , eif2ak3 , ptf1a , ipf1 , glis3 , rfx6 , slc2a2 , slc19a2 , foxp3 , gata6 , mnx1 , neurod1 , and hnf1b .
tndm is caused by defects associated with overexpression of paternally expressed genes in the imprinted region of chromosome 6q24 in 70% cases .
three reported defects include ( 1 ) paternal uniparental disomy of chromosome 6 ( upd6 ) ; ( 2 ) paternally inherited duplication of 6q24 ( duplication ) ; and ( 3 ) maternal hypomethylation at 6q24 .
about 26% of the patients contain mutations in kcnj11 , abcc8 , ins , or hnf1b .
the genetic etiology remains currently unknown in 40% of ndm cases . 
 in vitro and
clinical studies suggest that treatment with oral sulfonylurea can close katp channel and improve glycemic control and neuropsychological development [ 9 , 10 ] .
however , 10% of patients with kcnj11 and 15% abcc8 mutations fail to achieve glycemic control when insulin therapy is switched to oral sulfonylureas . therefore , molecular diagnosis is vital not only in accurate typing but also for better prognostication .
we summarized the clinical features , molecular typing , treatment , and 1- to 13-year follow - up of 25 cases of ndm in order to better understand the clinical treatment and prognosis .
the present study included 25 patients diagnosed with ndm including 18 pndm and 7 tndm from beijing children 's hospital , zhengzhou children 's hospital , and shanxi children 's hospital , from 2001 to 2013 .
diagnostic criteria for ndm   were as follows : ( 1 ) age at onset < 6 months ; ( 2 ) hyperglycemia sustained for 2 weeks ; ( 3 ) insulin dependence ; and ( 4 ) exclusion of hyperglycemia caused by stress and infection and drug therapies .
the family history of diabetes mellitus , especially glucose metabolism in parents , was recorded for every patient .
the self - reported frequency of severe hypoglycemia was recorded , and hba1c was measured at every visit . all the data between years 2012 and 2013 were analyzed .
patients aged below 18 months , showing normal blood glucose ( fasting glucose < 5.6  mmol / l , postprandial glucose < 7.8  mmol / l ) and hba1c ( < 6.0% ) without the need for insulin or oral hypoglycemic treatment , were defined as tndm .
patients aged more than 18 months and requiring insulin or oral hypoglycemic agents to maintain normal glucose were defined as pndm .
this study was approved by the ethics committee of beijing children 's hospital of capital medical university and all parents have signed the informed consent . upon ndm diagnosis ,
2  ml of blood samples was collected in ethylenediaminetetraacetic acid ( edta ) tubes from 25 patients and stored at 20c .
dna was extracted from peripheral blood leukocytes using kits ( qiagen , valencia , ca ) .
samples were tested for kcnj11 and abcc8 mutations using sanger sequencing annually , usually within 1 year after diagnosis .
sanger sequencing of the eif2ak3 was undertaken in one patient because of the presence of typical features of wolcott - rallison syndrome .
genes associated with ndm include kcnj11 , abcc8 , ins , gck , pdx1 , eif2ak3 , ptf1a , ipf1 , glis3 , rfx6 , slc2a2 , slc19a2 , foxp3 , gata6 , mnx1 , neurod1 , and hnf1b .
subsequently , sanger sequencing was used to validate the screened mutations and in parents for inherited or de novo mutations . confirmed mutations
were then searched in the human gene mutation database ( hgmd ) , dbsnpl38 , thousand genomes , and recent reviews .
for all mutations , software polyphen-2 was used to predict the pathogenicity ( http://genetics.bwh.harvard.edu/pph2/ ) .
microarray comparative genomic hybridization was performed in 5 tndm patients , in whom abcc8 and kcnj11 mutations were excluded .
we used 4  l10  l of patient dna for the assay , dna amplification , tagging , and hybridization according to the manufacturer 's protocol .
data analysis was performed using genomestudio version 2010.1 , karyostudio version 1.2 ( illumina , standard settings ) , and nexus copy number 5.0 ( biodiscovery , el segundo , ca , usa ) . the positive case was subjected to haplotype analysis using highly polymorphic short tandem repeat ( str ) markers that span both arms of chromosome 6 .
we recalled katp - pndm patients to switch from insulin injection to oral glyburide , usually within 18 months of diagnosis .
the transfer was carried out using a protocol that was similar to that described previously .
glyburide was started at a dose of 0.1  mg per kilogram twice daily and was increased daily by 0.2  mg per kilogram .
the dose of glyburide was increased until insulin independence was achieved or the dose was at least 0.8  mg per kilogram per day .
the change to sulfonylureas was considered to be successful if a patient was able to stop insulin treatment completely at any dose of glyburide and was deemed to be unsuccessful if insulin was still required with a dose of glyburide at least 0.8  mg per kilogram per day .
type 1 diabetic patients with age of onset between 6 months and 2 years were matched one - to - one with those of pndm ( 15 patients with recorded hba1c ) .
t1 dm patients hospitalized during the same period as pndm group with positive autoimmune antibody ( ica , gad , or iaa ) and comparable age , sex , duration of illness , and time of sample / data collection were selected as the control group .
the hba1c of the pndm group was tested at least 6 months after glyburide treatment .
statistical analysis was performed using chi - square test and student 's t - test using spss 19.0 ( spss inc . ,
continuous data were analyzed using student 's t - test , and categorical data was analyzed using chi - square test .
based on follow - up , 18 cases were typed as pndm ( 72.0% ) and 7 cases as tndm ( 28.0% ) .
no statistical differences were found between the two types with age of onset , birth weight , and dk / dka prevalence ( p > 0.05 ) ( see table 1 ) .
direct sequencing identified the most frequent mutations involving katp channel ( n = 6 ) including kcnj11 ( n = 5 ) and abcc8 mutation ( n = 1 ) , all of which were identified earlier .
the non - katp mutations ( n = 6 ) including ins , eif2ak3 ( n = 3 ) , glis3 , and slc19a2 were identified and five were confirmed as novel mutations . in the tndm , we found 2 cases harbouring abcc8 mutation and 1 case with upd6 .
all patients were treated with insulin initially . following stable glucose control with insulin , transition from insulin to oral sulfonylureas
finally , three cases ( 60% ) were successfully placed on glyburide ; one switched back to insulin as there was no response to glyburide ; one stopped oral glyburide because of serious gastrointestinal reactions ; and in another case glyburide was not tried because of loss of follow - up .
the mean hba1c of pndm during the last visit was 7.2  0.8% , which was similar to that of the infant - onset t1 dm group ( table 3 ) . except in 4 pndm cases ,
the patients who presented with convulsion at the onset showed no further convulsions . among patients with positive mutations , case 1 carrying kcnj11p.r201h mutation had congenital cataract .
glyburide therapy was also stopped in case 1 due to gastrointestinal reactions and it was switched back to regular insulin treatment with good glycemic control .
case 2 with kcnj11p.r201h mutation and case 3 with kcnj11p.g53s mutation achieved good blood glucose levels with no hypoglycemia with glyburide treatment .
case 5 bearing kcnj11p.e229k mutation was lost to follow - up 1 year after diagnosis , while he showed good response to initial insulin therapy but failed to undergo glyburide therapy later .
case 6 carrying the abcc8 p.r825w mutation failed to respond to glyburide ( 0.4  mg / kg / d ) , and the insulin dose was not reduced . during follow - up ,
insulin therapy once or twice daily was administered to the child depending on the blood glucose level .
case 4 ( kcnj11p.v59 m ) had intellectual and physical retardation without epilepsy and hence was diagnosed as idend syndrome .
this patient was given intermittent glyburide therapy ( following parental request ) but died of dka at 2 years of age in local hospital .
case 8 ( glis3 p.f857y ) had liver dysfunction but improved by following supportive therapy .
case 10 ( eif2ak3 p.c532stop ) was accompanied with intellectual and physical retardation , short stature , multiple skeletal dysplasia , and hypothyroidism , diagnosed as wolcott - rallison syndrome .
case 11 who manifested physical retardation and skeletal dysplasia showed compound heterozygous mutations in eif2ak3 .
case 14 died of dka at 7 months of age , several days after diagnosis .
the patient also showed weight loss and mental and physical retardation with negative results on genetic screening .
remission among tndm cases in our cohort was ascertained from 2 weeks to 1 year after diagnosis .
none of these patients showed any congenital abnormalities such as macroglossia , umbilical hernia , dysmorphic facial features , hematopoietic dysfunction or abnormal hearing , and heart , liver , or kidney function .
no specific features were observed in patient carrying abcc8 mutations and upd6 compared with the other tndm cases carrying negative genetic results .
the ndm patients generally presented with infection or decreased responsiveness at the onset , without the typical symptoms such as polyuria , polydipsia , polyphagia , and weight loss . in the present study ,
there were no differences between pndm and tndm patients in age of onset , birth - weight , and prevalence of dka , making it difficult to clinically distinguish between the two types .
the oldest child on follow - up was 5 years old with no recurrence of diabetes .
of the patients carrying kcnj11 and abcc8 mutations , there was developmental delay in one patient diagnosed as idend , including one with congenital cataract .
.   found that patients with mutations in katp channel subunit genes presented with developmental coordination disorders including visual - spatial dyspraxia and attention deficits but not developmental delay or epilepsy .
therefore , adults with a history of neonatal diabetes mellitus should be tested for neuropsychological dysfunction and developmental defects .
we identified six mutations in katp channel that were previously reported and six non - katp mutations in pndm cases , five of which were not identified until now , and found two katp mutations in tndm cases .
three cases were placed on glyburide therapy and 15 cases were on insulin therapy , with good glycemic control in most cases . in this study
we identified two cases of p.r201h mutations , which were the most common kcnj11 mutations without neurological abnormalities .
these mutations have been confirmed at cpg dinucleotide , resulting in decreased sensitivity of katp channels to atp .
glucose stimulation reduces insulin secretion , whereas sulfonylureas stimulate the secretion , explaining the rationale of glyburide therapy .
of the two cases , one was responsive to glyburide and the other presented with severe gastrointestinal reactions and parental worries about side effects and therefore discontinued despite recommendations to the contrary .
generally , patients with this mutation cause a triad of developmental delay , epilepsy , and neonatal diabetes ( dend syndrome ) , or without epilepsy ( intermediate dend ( idend ) syndrome ) .
the neurological symptoms are attributable to the presence of kir6.2 in nerve and brain tissue .
the patient with this mutation in the present study was successfully switched from insulin to glyburide , leading to hba1c level of 6.5% .
the fourth mutation was kcnj11p.e229k , which induces ndm without neurological manifestations but was lost to follow - up . as reported , p.e229 and p.g53 mutations cause both pndm and tndm [ 15 , 16 ] . in our study
the patient with kcnj11p.g53s underwent low dose glyburide ( 0.4  mg / kg / d ) , consistent with previous reports .
the mean effective dosage of glyburide is 0.5~0.6  mg / kg / d for sur1 mutations .
the parents stopped glyburide due to the side effects . at follow - up , the patient was on insulin therapy and refused to be treated with glyburide .
physicians must be cautious before using sulfonylureas as they are not licensed in children and may have side effects .
the glyburide transition can only be tried as an inpatient procedure to avoid diabetic ketoacidosis in those young children who may be unresponsive to glyburide and need careful monitoring .
parents who carried the same kcnj11 or abcc8 mutations showed no neonatal diabetes mellitus symptoms or past history of abnormal glucose metabolism .
the phenomenon suggests that katp mutations have variable clinical phenotypes , with the symptoms varying from tndm or pndm .
the mild and transient manifestations during neonatal period were missed , with possible risk of relapse in the future .
the glucose metabolism of parents carrying the same mutations as their children needs to be monitored .
the success rate of glyburide transition among cases with katp mutations of 60.0% in the present study is lower than that reported from other countries .
one reason for the low success rate could be the side effects of glyburide such as nausea and vomiting that affected its clinical application in infants in the present study .
diarrhea is the common reported side effects of glyburide but has not been seen in our patients .
the failed transitions due to side effects were not reported in other studies , suggesting that chinese children respond differently to glyburide compared with other ethnic groups .
case 1 manifesting mental and physical retardation , hypothyroidism , and multiple epiphyseal dysplasia was diagnosed with wrs , as reported by sang et al . .
sanger sequencing yielded a sole heterozygous mutation in this patient , which was inherited from father .
the loss of kinase in the catalytic domain resulted in a complete loss of function .
the italian pndm patient had heterozygous mutations in both kcnj11 and gck genes , suggesting that ndm may be a polygenic disease . however , no ngs was performed .
case 18 was also diagnosed as wrs carrying compound heterozygous mutations of eif2ak3 , inherited from father and mother , respectively , with one mutation reported and another deletion mutation resulting in a truncated protein .
ndm patients with glis3 gene mutation may also manifest congenital hypothyroidism , glaucoma , liver fibrosis , and polycystic kidney disease .
the patient with glis3 mutation showed hepatic dysfunction without liver fibrosis and polycystic kidney disease at onset but eventually died of liver and kidney failure 1 year later in a local hospital .
glis3 mutations have been reported to be autosomal recessive resulting in a variable clinical phenotype .
slc19a2 gene mutations causing ndm lead to the development of diabetes , deafness and megaloblastic anemia . this syndrome can be ameliorated by thiamine , so it is called thiamine - responsive megaloblastic anaemia ( trma ) .
our patient with slc19a2 mutation exhibited moderate normocytic anemia with no hearing abnormalities at diagnosis .
another mutation in a non - coding region or a large deletion may be found by sanger sequencing of the whole gene or with multiplex ligation probe amplification technology ( mlpa ) , respectively .
the classification of tndm and pndm should be reconsidered . a diagnosis of diabetes in parents who carried the mutations can not be excluded .
we need additional and longer follow - up of cases to establish a tndm diagnosis or pndm remission .
diarrhea is one of the manifestations of ipex ( immune dysregulation , polyendocrinopathy , enteropathy , and x - linked syndrome ) , which is caused by foxp3 mutation .
ipex may also present with autoimmune thyroid disease , exfoliative dermatitis , and sepsis caused by immune dysregulation .
the mutations in katp channel accounted for 33.3% of pndm , including 27.8% in kcnj11 mutations and 5.6% in abcc8 mutations , similar to the reported data .
we diagnosed two cases of wrs ( 11.2% of all pndm cases ) , which is now recognized as the most frequent cause of pndm in consanguineous children [ 23 , 24 ] .
in addition , rare gene mutations ( glis3 , slc19a2 , etc . ) also were associated with specific clinical manifestations .
therefore , in view of the high mutation rate of kcnj11 in pndm and high correlation between genotype and phenotype , we suggest screening for kcnj11 mutations first in ndm cases , followed by abcc8 screen and genes associated with specific complications .
pndm children on insulin and glyburide therapy were found to have good glycemic control ( mean hba1c 7.2% ) during follow - up , which is similar to infantile onset t1 dm group .
it was probably associated with younger age at onset , need for a lower insulin dose , and better residual pancreatic  cell function .
the onset age of pndm and t1 dm is usually different , although the duration of illness is comparable , which is the most important factor affecting the hba1c level .
the syndrome is associated with giant tongue , umbilical hernia , facial deformity , kidney abnormalities , congenital heart disease , hypothyroidism , and intrauterine growth retardation ( > 95% ) .
array - cgh could be used to detect upd6 and duplication simultaneously , so it may be a cost - effective genetic analysis method for tndm cases . in our study , all the tndm cases did not exhibit the specific manifestations mentioned above .
recurrence of tndm resulting from kcnj11 , abcc8 , and 6q24 mutations might respond to treatment with sulfonylurea [ 3 , 25 ] .
it should be noted that the pathophysiological mechanism of 6q24 mutations is not clear and thus the most appropriate treatment remains unclear . in the present study ,
the genetic etiology could not be determined in 50% of pndm and 57% of tndm cases .
however , no maternal hypomethylation at chr6q24 was detected , which may have affected the mutational analysis of the tndm cohort .
most ndm patients achieved good glycemic control ( hba1c < 7.5% ) and there was no significant difference when compared to infantile onset t1 dm .
 
 aims . to study the clinical features , genetic etiology , and the correlation between phenotype and genotype of neonatal diabetes 
 mellitus ( ndm ) in chinese patients . 
 methods . 
 we reviewed the medical records of 25 ndm patients along with their follow - up details . 
 molecular genetic analysis was performed . 
 we compared the hba1c levels between pndm group and infantile - onset t1 dm patients . 
 results . of 25 ndm patients , 
 18 ( 72.0% ) were pndm and 7 ( 28.0% ) were tndm . among 18 pndm cases , 6 ( 33.3% ) 
 had known katp channel mutations ( katp - pndm ) . 
 there were six non - katp mutations , five novel mutations , including ins , eif2ak3 ( n = 2 ) , glis3 , and slc19a2 , one known eif2ak3 mutation . 
 there are two abcc8 mutations in tndm cases and one paternal upd6q24 . 
 five of the six katp - pndm patients were tried for glyburide transition , and 3 were successfully switched to glyburide . 
 mean hba1c of pndm was not significantly different from infantile onset t1 
 dm ( 7.2% versus 7.4% , p = 0.41 ) . conclusion . 
 pndm accounted for 72% of ndm patients . 
 about one - third of pndm and tndm patients had katp mutations . 
 the genetic etiology could be determined in 50% of pndm and 43% of tndm cases . 
 pndm patients achieved good glycemic control with insulin or glyburide therapy . 
 the etiology of ndm suggests polygenic inheritance . 
1. Introduction
2. Subjects and Methods
3. Results
4. Discussion