hepatocyte nuclear factor 4a ( hnf4a ) is an orphan member of the nuclear receptor family of transcription factors ( 1 ) .
it is expressed primarily in the liver , gut , kidney and pancreas and has important role during embryonic life in pancreatic development and maintaining of  cell function ( 2 ) .
indeed , knockout studies found that hnf4a/ mice revealed impaired gastrulation and died around day e9 ( 3 ) .
the hnf4a protein consists of five functional domains : an n - terminal activation function ( af-1 , also referred to as a / b domain ) , two zinc fingers responsible for dna binding ( c domain ) , a potential ligand binding domain ( e domain ) and the f domain ( 1 ) . hnf4a gene ( ensg00000101076 ) has many transcripts .
the enst00000316099 transcript is considered the canonical transcript that codes the isoform hnf4-alpha-1 with 474 amino - acids ( p41235 - 1 ) .
mutations in hnf4a gene are responsible of mody-1 ( maturity - onset diabetes of the young type 1 ) ( omim#125850 ) and neonatal hyperinsulinism hypoglycemia ( 5 , 6 ) . to date , about 103 hnf4a inactivating mutations were reported in 173 mody families ( 7 ) .
some hnf4a variants were found to be associated with type 2 diabetes ( t2d ) ( 8) .
t2d is a chronic disorder of glucose metabolism , characterized by a state of hyperglycemia due to defective insulin secretion , peripheral insulin resistance and increased hepatic glucose production ( 9 ) .
genetic and environmental factors , contribute to the appearance of t2d ( 10 ) . whereas , mody is a dominantly inherited form of early - onset type 2 diabetes ( t2d ) , generally diagnosed in childhood , adolescence or young adulthood ( 11 ) .
it is a rare monogenic disease caused by primary defects of insulin secretion with a lack of auto - antibodies against the pancreatic cells and is rarely associated with obesity ( 11 ) .
the nm_000457.4 : c.1387a > g or hnf4a - p.i463v mutation ( rs147638455 or cm993900 ) , also known as p.i431v and p.i453v depending on the corresponding gene s isoform , was previously found in a caucasian and a tunisian families suspected of mody ( 12 , 13 ) .
but , it remains unclear whether this variant is a mutation responsible for mody-1 or a polymorphism . in the case of a molecular diagnosis of mody-1 ,
finding hnf4a - p.i463v would be confusing : should we consider it as a mutation ?
in this report , we present a simple and easy protocol to screen hnf4a - p.i463v variant .
we also assessed its frequency in tunisian north - african population and discussed its probable effect .
two hundred anonymous unrelated healthy controls were recruited in 2013 from the blood transfusion center of sousse ( tunisia ) .
they were examined by the physician of the center and were found not diabetic and did not present any other disease .
the study was carried out after taking informed written consent from all individuals and after approval of the ethics committee of farhat hached university hospital .
first , hnf4a exon 10 including exon - intron boundaries was amplified by pcr using the following primers f : 5 ttt act ccc aca aag gct gg 3 and r : 5 atc acc agg tgc tct ctt ag 3. pcr was performed in a 50 l volume containing 50 ng of genomic dna , 20 pmol of each primer and 1 u of recombinant dna polymerase ( invitrogen , carlsbad , ca , usa ) .
fifteen l of pcr product were mixed with 2l bsa , 2l buffer , 5.75 l sterilized water and digested by 2.5 u of foki restriction enzyme ( takara bio inc , otsu , shiga , japan).the digested pcr products were then separated on 2.5% agarose gel .
thus , the wild fragment ( 266 bp ) is cut into 164 bp and102 bp fragments .
finally , in case of mutation detection , direct sequencing was used to confirm it .
the p.i463v substitution was studied in - silico to predict the putative effect on protein function .
we performed in - silico analysis using two different types of software : polyphen2 ( http://genetics.bwh.harvard.edu/pph2/index.shtml ) and mutation t@sting ( http://www.mutationtaster.org/ ) .
these bioinformatics tools enable high - throughput prediction of the potential impact of residue changes and large - scale polymorphism analyses .
polyphen and mutation t@sting predict possible impact of an amino acid substitution on the structure and function of a human protein using straight forward physical and comparative considerations ( 14 , 15 ) .
two hundred anonymous unrelated healthy controls were recruited in 2013 from the blood transfusion center of sousse ( tunisia ) .
they were examined by the physician of the center and were found not diabetic and did not present any other disease .
the study was carried out after taking informed written consent from all individuals and after approval of the ethics committee of farhat hached university hospital .
first , hnf4a exon 10 including exon - intron boundaries was amplified by pcr using the following primers f : 5 ttt act ccc aca aag gct gg 3 and r : 5 atc acc agg tgc tct ctt ag 3. pcr was performed in a 50 l volume containing 50 ng of genomic dna , 20 pmol of each primer and 1 u of recombinant dna polymerase ( invitrogen , carlsbad , ca , usa ) .
fifteen l of pcr product were mixed with 2l bsa , 2l buffer , 5.75 l sterilized water and digested by 2.5 u of foki restriction enzyme ( takara bio inc , otsu , shiga , japan).the digested pcr products were then separated on 2.5% agarose gel .
thus , the wild fragment ( 266 bp ) is cut into 164 bp and102 bp fragments .
finally , in case of mutation detection , direct sequencing was used to confirm it .
the p.i463v substitution was studied in - silico to predict the putative effect on protein function .
we performed in - silico analysis using two different types of software : polyphen2 ( http://genetics.bwh.harvard.edu/pph2/index.shtml ) and mutation t@sting ( http://www.mutationtaster.org/ ) .
these bioinformatics tools enable high - throughput prediction of the potential impact of residue changes and large - scale polymorphism analyses .
polyphen and mutation t@sting predict possible impact of an amino acid substitution on the structure and function of a human protein using straight forward physical and comparative considerations ( 14 , 15 ) .
the assessment of hnf4a - p.i463v variant in 200 healthy tunisian people by rlfp - pcr method found that 3 controls ( 1.5% ) were carriers of this mutation .
hnf4a - p.i463v variant detected after sequencing and enzymatic digestion by foki restriction enzyme a : gel electrophoresis of hnf4a exon 10 ( 266 bp ) before digestion by foki restriction enzyme in 9 controls .
b : gel electrophoresis of hnf4a exon 10 after digestion by foki restriction enzyme into 2 fragments ( 164 and 102 bp ) in 7 controls .
c : chromatograph of a part of hnf4a exon 10 sequence showing the c.1387a > g / p.i463v mutation the tunisian population of this study is in hardy - weinberg equilibrium ( table 1 ) .
the frequency of hnf4a - p.i463v is rare in the other populations . indeed , this variant is more frequent in the tunisian population than the european and african american populations .
rs147638455 frequency in different populations exome sequencing project , csagilent ( dbsnp 138 ) , : the tunisian population is in hardy - weinberg equilibrium ( p=0.91 > 0.05 ) polyphen2 predicted that this nucleotide change is a  polymorphism  with a score of 0.144 [ 01 ] .
while , the mutation t@sting software indicated that this variant could be responsible of a probably affected protein .
the assessment of hnf4a - p.i463v variant in 200 healthy tunisian people by rlfp - pcr method found that 3 controls ( 1.5% ) were carriers of this mutation .
hnf4a - p.i463v variant detected after sequencing and enzymatic digestion by foki restriction enzyme a : gel electrophoresis of hnf4a exon 10 ( 266 bp ) before digestion by foki restriction enzyme in 9 controls .
b : gel electrophoresis of hnf4a exon 10 after digestion by foki restriction enzyme into 2 fragments ( 164 and 102 bp ) in 7 controls .
c : chromatograph of a part of hnf4a exon 10 sequence showing the c.1387a > g / p.i463v mutation
the tunisian population of this study is in hardy - weinberg equilibrium ( table 1 ) .
indeed , this variant is more frequent in the tunisian population than the european and african american populations .
rs147638455 frequency in different populations exome sequencing project , csagilent ( dbsnp 138 ) , : the tunisian population is in hardy - weinberg equilibrium ( p=0.91 > 0.05 )
polyphen2 predicted that this nucleotide change is a  polymorphism  with a score of 0.144 [ 01 ] . while , the mutation t@sting software indicated that this variant could be responsible of a probably affected protein .
performing a molecular diagnosis of monogenic diabetes is not an easy task because of its clinical and genetic heterogeneity ( 16 ) .
some genetic variants could also be problematic such as hnf4a - p.i463v . in an earlier study
, we found that hnf4a - p.i463v cosegregated with diabetes phenotype in a tunisian family ( fig . 2 and table 2 ) .
it was present in three patients from this family and was absent in two other non - affected relatives ( 13 ) .
contrariwise , this variant did nt show a perfect cosegregation in a caucasian family ( 12 ) .
hitherto , there is not enough information in the literature about hnf4a - p.i463v variant ( rs147638455 ) .
it is not clear whether it is a mutation directly causing mody-1 or just a non - synonymous polymorphism with low effect .
the clinical characteristics of the reported hnf4a - p.i463v carriers na : not applicable , nf : not found , oha = oral hypoglycemic agents , ins : insulin the diabetic caucasian and tunisian families having hnf4a - p.i463v variant filled and open symbols represent diabetic subjects and normal glucose tolerance individuals , respectively .
below the numbers , it is the genotype at codon i463v : n , normal allele ( isoleucine ) ; m , mutant allele ( valine ) . below the genotype
is the diagnostic age of diabetes for affected members and age at examination , followed by the treatment for diabetes ( oha : oral hypoglycemic agents and ins : insulin ) .
hnf4a - p.i463v a variant with interesting characteristics this variant occurs in a conserved region across species ( phylop score = 4.56 [ 14.1 ; 6.4 ] ) and is localized in the f domain of hnf4a ( fig .
3 ) . this domain plays a role in the protein transactivation activity ( 17 ) .
isoleucine amino - acid in position 463 is conserved across species a portion of hnf4a exon 10 amino - acid sequence is compared between various species .
the human isoleucine at codon 463 and isoleucine of other species are framed in a red box .
the species are annotated in bold and the ncbi accession numbers in blue hnf4a - p.i463v variant could not be responsible for mody-1 using a simple protocol of screening for hnf4a - p.i463v variant ( rs147638455 ) , we detected it in three healthy controls out of 200 tunisian unrelated healthy individuals ( 400 chromosomes ) .
indeed , it is an unexpected high carrier frequency ( 1.5% ) in this sample compared with the other populations ( table 1 ) .
the healthy controls carriers of rs147638455 in our population are aged 20 , 26 and 37 years ( table 2 ) .
even if we omit the 20 year - old control individual , the rs147638455 frequency would remain high for a mutation ( 1% ) .
in other ethnic populations , rs14763845 was rarely found in healthy individuals ( table 1 ) . if we suppose that this variant is responsible for mody-1 , which is a rare monogenic disease , tunisian frequency , argues against this hypothesis . on the other hand ,
the in silico analysis using mutation t@ster and polyphen2 showed conflicting results . in parallel , physicochemical difference between isoleucine and valine amino - acids is small ( grantham distance = 29 [ 0215 ] ) .
in addition to the absence of a cosegregation of hnf4a - p.i463v variant in the caucasian family , the tunisian family suspected of mody had a mean age of diabetes diagnosis of 38  10.58 years old ( 13 ) ( table 2 and fig .
otherwise , a previous study showed that patients with mutations in hnf4a exons 9 and 10 developed diabetes later than those with mutations in exons 28 did(4 ) .
so , mutations in exons 9 and 10 could not be responsible for mody-1 ( 1 ) .
speculations about the effect of this variant and its implication in type 2 diabetes hnf4a - p.i463v could act in an interactive polymorphisms network leading to t2d . at that time
, the relatively young age of our healthy carriers ( 20 , 26 and 37 years old ) does not definitely mean that they will not present t2d later .
a similar missense mutation in the f domain ( hnf4a - p.v393i localized in exon 9 ) was found in a french diabetic family diagnosed at the age of 45 years .
hnf4a - p.v393i was associated with a marked reduction of transactivation activity responsible for impaired insulin secretion and co - segregated with diabetes ( 18 ) .
it is of note that some non diabetic individuals aged between 30 and 40 year - old were carriers of hnf4a - p.v393i ( 18 ) .
indeed , most of the patients that carry hnf4a - p.i463v variant are over 30 years ( table 2 )
association and functional studies should be carried out , to assess the exact effect of hnf4a - p.i463v variant .
meantime , we recommend to do not consider hnf4a - p.i463v as a mutation in case of molecular diagnostic of mody .
this recommendation is based on the high frequency of this variant in the tunisian population , its presence in healthy persons and the conflicting results of the in silico analysis .
we can speculate that hnf4a - p.i463v is a polymorphism with possible role in t2d and not a causal mutation implicated directly in mody-1 .
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 : hnf4a - p.i463vvariant , reported previously in two distinct families suspected of mody-1 , is assessed in this report to determine whether it is a mutation or a polymorphism ( frequency > 1%).methods:200 tunisian healthy people were screened for the presence of hnf4a - p.i463v variant , using rflp - pcr technique and sequencing . 
 then , the frequency of this variant was estimated in the tunisian population and compared to other populations registered in genetic databases . 
 we also performed in - silico analysis using polyphen2 and mutation t@sting softwares to assess the probable effect of hnf4a - p.i463v variant.results:hnf4a-p.i463v had a rare frequency in different populations and was found in 3 control subjects ( 1.5% ) of the studied population . 
 polyphen2 predicted that it is a polymorphism , whereas mutation t@sting suggested a probably affected mutant protein.conclusion:hnf4a-p.i463v has a relatively high frequency ( > 1% ) in our control cohort . 
 it is also present in different ethnicities and in- silico analysis showed conflicting results . for these reasons , hnf4a - p.i463v 
 should not be considered as a mutation responsible for mody-1 . 
Introduction
Materials and Methods
DNA samples
HNF4A -p.I463V mutation analysis
Statistical analysis
Prediction of p.I463V-HNF4A mutation effect in-silico
Results
Carrier Frequency of p.I463V
Comparison with other populations
Results of in Silico Analysis
Discussion
Conclusion
Ethical considerations