maturity - onset diabetes of the young ( mody ) was distinguished from other types of diabetes mellitus in 1970 ( 1 ) .
it is clinically characterized by nonketotic diabetes mellitus , a primary defect in the function of the beta cells of the pancreas , an onset before the age of 25 yr ( usually in childhood or adolescence ) , and an autosomal dominant mode of inheritance ( 2 ) .
mody seems to be an attractive model for genetic studies because its well - defined mode of autosomal dominant inheritance with high penetrance , and its early onset age that aids the collection of multigenerational pedigrees ( 3 ) .
the molecular genetic basis of mody started in the 1990s ( 46 ) , showed that mody is a heterogenous group of monogenic disorders .
to date , online mendelian inheritance in man ( omim ) has listed mody1 - 11 based on implicated genes ( table 1 ) ( 7 ) . genetic classification and clinical phenotypes of the mody subtypes adapted from attiya
et al ( 41 ) mody - related genes that have been most commonly studied are the hepatic nuclear factor 4 alpha ( hnf4 ) ( mody1 ) , glucokinase ( gck ) ( mody2 ) , the hepatic nuclear factor 1 alpha ( hnf1 ) ( mody3 ) , the insulin promoter factor-1 ( ipf1 ) ( mody4 ) and the hepatic nuclear factor 1 beta ( hnf1 ) ( mody5 )
for example , around 32% , 52% , 10% and 6% of cases in the uk were due to gck , hnf1 , hnf4 , and hnf1 , respectively ( 8) . to adding the heterogeneity of mody , diverse mutations were reported in each pathogenic gene . for gck only , more than 600 mutations have been described so far , leading to both hypoglycemia and hyperglycemia ( 9 ) . furthermore
, 1645% of the cases of mody can not be sorted into these 11 subtypes , and be named mody - x .
more studied are needed to discover the unknown mody locus or loci ( 10 ) .
population studies revealed that heterogeneity also exists within mody patients of different ethnic groups ( 11 ) .
for example , mody2 ( gck ) and mody3 ( hnf-1a ) were found to account for more than 80% of cases in a cohort of u.k .
however , in hong kong chinese , these two forms of mody were found to be responsible for a mere 4% and 5% , respectively ( 13 ) . except for the subtype heterogeneity ,
the mutations of the same mody subtype that has been reported in different populations are usually different and hard to be confirmed ( 11 ) .
to help the overall understanding of mody - related pathologic mutations , we studied a large chinese mody family .
we use whole genome scan and candidate gene sequencing to identify the mody subtype of the family , and to find the related gene mutation .
a large han pedigree in 2012 in shandong province , northern china , was identified through a proband with typical mody features ( fig .
1 ) . pedigree of the mody family from changqing , shandong , china the pattern of inheritance within the family is consistent with autosomal - dominant inheritance . in total , 9 individuals in this three - generation family met mody diagnostic criteria ( 14 ) and showed a broad spectrum of clinical diabetic phenotypes .
this study obtained signed informed consent from 9 affected and 17 unaffected family members or their guardians , and 1000 adult voluntary blood donors .
this study was approved by the ethics committee of the institute of basic medicine of shandong academy of medical sciences .
peripheral venous blood samples were collected from 9 mody patients in the family ( 2 males and 7 females , age 1554 ) , 17 unaffected family members ( 8 males and 9 females , age 777 ) and 1000 apparently healthy blood donors randomly selected from the blood center of shandong province ( 702 males and 298 females , aged 1755 ) .
genomic dna was extracted by a modified phenol - chloroform method as described before ( 15 ) .
the pooled dna samples of ( i ) mody patients in the family ( n=9 ) , ( ii ) unaffected family members ( n=17 ) , and ( iii ) unrelated population controls ( n=1000 ) , were obtained by combining equal amounts of dna from each individual ( 16 ) .
a panel of 400 microsatellite marker loci was genotyped in three pooled dna samples , using abi prism linkage mapping set md-10 ( abi ) .
the set has fluorescence - labeled pcr primer pairs for 400 highly polymorphic dinucleotide - repeat microsatellite markers chosen from the geneth on human genetic linkage map on both autosomal and sex chromosomes , with an average spacing of 10 cm ( 17 ) .
polymerase chain reaction ( pcr ) , electrophoresis and genotyping analysis were as described before ( 15 ) .
fifteen-l pcr mixture contained 20 ng of genomic dna , 1.5 mm mgci2 , 10x reaction buffer , 0.2 mm of each dntp , 0.3 m of each primer , and 1u of taq polymerase .
amplification was performed on a gene amp 9700 thermo cycler ( applied bio systems ) with the following parameters : 95 c for 12 min initial denaturation , followed by 94 c 15 sec , 55 c 15 sec , 72 c 30 sec , 10 cycles ; then 89 c 15 sec , 55 c 15 sec , 72 c 30 sec , 20 cycles , then 72 c 10 min , and 4 c hold .
electrophoresis was carried out on abi prism 3100 avant genetic analyzer , by mixing up to 9 pcr products simultaneously , which had no overlap of products of the same size range labelled with the same dye .
the predicted allele frequencies of microsatellites in each dna pool were calculated as allele frequency = fluorescence peak height of each allele / the sum of fluorescence peak height of all allele at the marker .
allele frequency was converted into allele counts and the statistical significance was calculated with clump24 , a dos executable with c source code based on monte carlo method for assessing significance of case - control association studies with multi - allelic markers ( http://www.davecurtis.net/dcurtis/software.html ) ( 18 ) . to reduce false positive error ,
primers for nine exons of hnf-1 ( listed in table 2 ) were designed using primer premier 5.0 software ( http://www.premierbiosoft.com ) .
pcr in a 50 l volume was performed with 5 l genomic dna ( 20 ng/l ) , 5 l of each primer ( 5 m ) and 25 l of multiplex pcr mix ( takara ) was performed on a geneamp9700 thermo cycler ( abi ) with the following parameters : 94 c for 5 min initial denaturation , followed by 94 c 30 sec , 59 c 30 sec , 72 c 60 sec , 45 cycles ; then 72 c 7 min , and 4 c hold . after spin column purification
, the reaction products was sequenced using bigdye terminator v3.1 cycle sequencing kit ( abi ) .
the purified labeled dna fragments were sequenced by applied bio systems 3130 genetic analyzer ( abi ) and analyzed by sequencing analysis software 5.2 ( abi ) .
a large han pedigree in 2012 in shandong province , northern china , was identified through a proband with typical mody features ( fig .
1 ) . pedigree of the mody family from changqing , shandong , china the pattern of inheritance within the family is consistent with autosomal - dominant inheritance . in total , 9 individuals in this three - generation family met mody diagnostic criteria ( 14 ) and showed a broad spectrum of clinical diabetic phenotypes .
this study obtained signed informed consent from 9 affected and 17 unaffected family members or their guardians , and 1000 adult voluntary blood donors .
this study was approved by the ethics committee of the institute of basic medicine of shandong academy of medical sciences .
peripheral venous blood samples were collected from 9 mody patients in the family ( 2 males and 7 females , age 1554 ) , 17 unaffected family members ( 8 males and 9 females , age 777 ) and 1000 apparently healthy blood donors randomly selected from the blood center of shandong province ( 702 males and 298 females , aged 1755 ) .
genomic dna was extracted by a modified phenol - chloroform method as described before ( 15 ) .
the pooled dna samples of ( i ) mody patients in the family ( n=9 ) , ( ii ) unaffected family members ( n=17 ) , and ( iii ) unrelated population controls ( n=1000 ) , were obtained by combining equal amounts of dna from each individual ( 16 ) .
a panel of 400 microsatellite marker loci was genotyped in three pooled dna samples , using abi prism linkage mapping set md-10 ( abi ) .
the set has fluorescence - labeled pcr primer pairs for 400 highly polymorphic dinucleotide - repeat microsatellite markers chosen from the geneth on human genetic linkage map on both autosomal and sex chromosomes , with an average spacing of 10 cm ( 17 ) .
polymerase chain reaction ( pcr ) , electrophoresis and genotyping analysis were as described before ( 15 ) .
fifteen-l pcr mixture contained 20 ng of genomic dna , 1.5 mm mgci2 , 10x reaction buffer , 0.2 mm of each dntp , 0.3 m of each primer , and 1u of taq polymerase .
amplification was performed on a gene amp 9700 thermo cycler ( applied bio systems ) with the following parameters : 95 c for 12 min initial denaturation , followed by 94 c 15 sec , 55 c 15 sec , 72 c 30 sec , 10 cycles ; then 89 c 15 sec , 55 c 15 sec , 72 c 30 sec , 20 cycles , then 72 c 10 min , and 4 c hold .
electrophoresis was carried out on abi prism 3100 avant genetic analyzer , by mixing up to 9 pcr products simultaneously , which had no overlap of products of the same size range labelled with the same dye .
the predicted allele frequencies of microsatellites in each dna pool were calculated as allele frequency = fluorescence peak height of each allele / the sum of fluorescence peak height of all allele at the marker .
allele frequency was converted into allele counts and the statistical significance was calculated with clump24 , a dos executable with c source code based on monte carlo method for assessing significance of case - control association studies with multi - allelic markers ( http://www.davecurtis.net/dcurtis/software.html ) ( 18 ) . to reduce false positive error ,
primers for nine exons of hnf-1 ( listed in table 2 ) were designed using primer premier 5.0 software ( http://www.premierbiosoft.com ) .
pcr in a 50 l volume was performed with 5 l genomic dna ( 20 ng/l ) , 5 l of each primer ( 5 m ) and 25 l of multiplex pcr mix ( takara ) was performed on a geneamp9700 thermo cycler ( abi ) with the following parameters : 94 c for 5 min initial denaturation , followed by 94 c 30 sec , 59 c 30 sec , 72 c 60 sec , 45 cycles ; then 72 c 7 min , and 4 c hold . after spin column purification
, the reaction products was sequenced using bigdye terminator v3.1 cycle sequencing kit ( abi ) .
the purified labeled dna fragments were sequenced by applied bio systems 3130 genetic analyzer ( abi ) and analyzed by sequencing analysis software 5.2 ( abi ) .
of all the 400 microsatellite marker loci in abi prism linkage mapping set md-10 ( abi ) , d17s798 on chromosome 17q11.2 was related with mody in the family .
allele frequencies for d17s798 were found to differ significantly ( p<0.001 ) between the affected and unaffected family members , the affected and the healthy controls , but not between unaffected family members and the healthy controls ( p=0.94 ) ( fig .
no microsatellite markers on sex chromosomes showed significant differences for frequency between any of the dna pooling samples , indicating an autosomal dominant inheritance that is one of the mody criterions .
allele frequency estimates for d17s798 close to d17s798 ( 17q11.2 ) , there is a known mody - related gene on 17q12 , hepatocyte nuclear factor-1 ( hnf-1 ) .
the differences of allele frequencies for the d17s798 mentioned above implicated that the affected family members can be sorted as the mody5 subtype .
a single nucleotide variant ( snv ) of c77 t in the 5-terminal non - coding region of exon 1 of hnf-1 was related to mody5 in the proband by sequencing and alignment analysis ( fig .
of all the 400 microsatellite marker loci in abi prism linkage mapping set md-10 ( abi ) , d17s798 on chromosome 17q11.2 was related with mody in the family .
allele frequencies for d17s798 were found to differ significantly ( p<0.001 ) between the affected and unaffected family members , the affected and the healthy controls , but not between unaffected family members and the healthy controls ( p=0.94 ) ( fig .
no microsatellite markers on sex chromosomes showed significant differences for frequency between any of the dna pooling samples , indicating an autosomal dominant inheritance that is one of the mody criterions .
close to d17s798 ( 17q11.2 ) , there is a known mody - related gene on 17q12 , hepatocyte nuclear factor-1 ( hnf-1 ) .
the differences of allele frequencies for the d17s798 mentioned above implicated that the affected family members can be sorted as the mody5 subtype .
a single nucleotide variant ( snv ) of c77 t in the 5-terminal non - coding region of exon 1 of hnf-1 was related to mody5 in the proband by sequencing and alignment analysis ( fig .
in the current study , allelic frequency of marker d17s798 on chromosome 17q11.2 was found to be related to mody in the chinese family .
the known mody - related gene near d17s798 is hnf-1 , implicating the patients in the family were mody5 subtype .
further sequencing and alignment analysis found that a single nucleotide variant ( snv ) of c77 t in the 5-terminal non - coding region of exon 1 of hnf-1 was related to mody5 in the proband .
mody is an entity of several monogenic disorders , and 11 genes have been associated to different mody subtypes : mody1-hnf4 , mody2-gck , mody3-hnf1 , mody4-pdx1 , mody5-hnf1 , mody6-neurod1 , mody7-klf11 , mody8-cel , mody9-pax4 , mody10-ins , mody11-blk ( 7 ) .
another research interest in the mody studies is heterogeneous across populations . the relative prevalence of each sub - types varied ( 21 , 22 ) . in european populations , mody2 ( the most prevalent form in french families ) ( 21 ) and mody3 ( the most prevalent form in british families ) ( 22 )
kindred clinically diagnosed as mody , a relative prevalence of 36% of mody3 , 10% of mody2 and 3% of mody1 were reported ( 23 ) . in spain , relative frequencies in the mody group were 80% mody2 , 8.5% mody3 and 1% mody5 ( 24 ) .
the other mody subtypes are rarely described in european studies . however , the studies in other continents reported that mody2 and mody3 are less prevalent .
for example , mody2 and mody3 mutations were accounted for 13% and 8.7% in 23 brazilian families only ( 25 ) .
similarly , in asia , mody2 and mody3 do not explain the majority of mody cases ( 2628 ) .
found in 79 mody japanese patients , type 2 and type 3 accounts for about 36.7% ( 11 plus 18 ) .
next most prevalent were mody 5 ( 6 patients ) and mody 1 ( 3 patients ) ( 26 ) . in a cohort of 27 mody korean patients and 17 patients with early onset type 2
diabetes , mody2 and mody 3 variants were found in only 3 patients ( including 1 mody patient ) ( 27 ) . in china particularly , a comprehensive study on 146 hong kong chinese mody families found 13 families had mody3 mutations ( 9% ) , 2 had mody2 mutations ( 1% ) .
the authors concluded that the majority of chinese mody patients are due to yet unknown genes ( 29 ) .
wang et al . screened in 154 unrelated probands from early onset and multiple affected diabetes chinese pedigrees and found only two patients carried mutation of hnf-1 gene ( 30 ) . here , we reported a large mody5 family found in the northern china , as a supplement to the above two chinese study which are based in the southern china .
hnf-1 gene ( gene i d : 6928 ) is located in chromosome 17:37686432 - 37745247 , encoded a protein of 557 amino acids in length ( http://asia.ensembl.org/ ) .
missense mutations that caused protein sequence changes were reported in different populations , e.g. m160v in australian ( 31 ) , p239r in romanian ( 34 ) , l264s in japanese patients ( 32 ) , p159l in korean patients ( 35 ) and s36f in chinese patients ( 30 ) . a frame - shift mutation , c.c1304del was found in italy patients , which resulted in a truncated protein ( 36 ) . monoallelic loss of the entire hnf1b gene were reported in merman patients who showed more severe clinical symptoms other than diabetes , such as pancreas dysplasia , exocrine insufficiency , genital defects and mental retardation ( 37 ) . in the systematic review of hnf-1 anomalies related to mody5 ( 38 ) , the detection rate of hnf-1 anomalies was 1.4% among mody cases ( 1.4% ) and 41.2% among mody cases with renal structure anomalies .
mutations were strikingly located within the dna binding domain and spread among exons of the dna binding domain with exons 2 and 4 were the hottest spots . however , in our mody family an exon 1 mutation was implicated .
this novel snv is located in the 5-terminal non - coding region of exon 1 .
the function of the 5 non - coding region of eukaryotic mrna and its binding proteins has not been extensively studied .
generally , the 5 non - coding region plays a crucial role in the protein translation by forming secondary structure and providing binding sites for the ribosome and translation factors ( 39 ) .
therefore , we hypothesize that the change from a  methylateable  base c to a  non - methylateable
 base t in the 5non - coding region of hnf-1 may change transcription levels by altering the mrna secondary structure and influence the binding sites for ribosome and/or translation factors .
the svp of c77 t in the 5non - coding region of hnf-1 was related to mody5 in the chinese family studied .
previously detected mutations mainly indicated pathology resulting from the relation between hnf-1 protein structure / function and primary insulin secretory defects .
as this novel mutation is in a non - coding region of the gene it suggests that regulation of the gene may be worthy of attention in studying the pathological mechanism of mody or diabetes .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
 background : maturity - onset diabetes of the young ( mody ) is a heterogeneous entity of monogenic disorders characterized by autosomal dominant inheritance . 
 eleven genes were related , including hnf4 , gck , hnf1 , ipf1 , and hnf-1 , and various mutations are being reported.methods:to help the overall understanding of mody - related pathologic mutations , we studied a large mody family found in 2012 , in shandong , china , which contained 9 patients over 3 generations . 
 dna was extracted from the periphery blood samples of ( i ) 9 affected members , ( ii ) 17 unaffected members , and ( iii ) 1000 healthy controls . 
 three pooled samples were obtained by mixing equal quantity of dna of each individual within the each group . 
 totally 400 microsatellite markers across the whole genome were genotyped by capillary electrophoresis . 
 the known mody - related gene near the identified marker was sequenced to look for putative risk variants.results:allelic frequency of marker d17s798 on chromosome 17q11.2 were significantly different ( p<0.001 ) between the affected vs. unaffected members and the affected vs. healthy controls , but not between the unaffected members vs. healthy controls . 
 mody5-related gene , hepatocyte nuclear factor-1 ( hnf-1 ) on 17q12 near d17s798 became the candidate gene . 
 a single nucleotide variant ( snv ) of c77 t in the non - coding area of exon 1 of hnf-1 was found to be related to mody5.conclusion:this novel snv of hnf-1 contributes to the diabetes development in the family through regulating gene expression most likely . 
 the findings help presymptomatic diagnosis , and imply that mutations in the non - coding areas , as well as in the exons , play roles in the etiology of mody . 
Introduction
Materials and Methods
Pedigree assessment
DNA extraction and sample pooling
Genotyping and statistics
Sequencing and alignment for HNF-1
Results
Genotyping of 400 microsatellite markers in three pooled DNA samples revealed D17S798 as a positive marker to MODY
The mutation of C77T in exon 1 of HNF-1 gene was associated with MODY5 in the family
Discussion
Conclusion
Ethical considerations