cystinuria is an autosomal recessive disorder characterized by the abnormal urinary excretion of cysteine and the dibasic amino acids in the renal tubule and epithelial cells of small intestine .
two genes including slc3a1 and slc7a9 are associated with cystinuria and two different types of cystinuria have been known according to genetic defects .
type i of cystinuria is caused by mutations in slc3a1 , an amino acid transporter gene located on chromosome 2 ( 2p16.3 - 21 ) , and consists of 10 exons ranging in span from 120 to 461 bp which encodes the b transporter - related protein ( rbat ) .
this protein creates the heavy chain of the renal cystine transport system ( rbat / b at ) . due to its biological functions , mutations in slc3a1/rbat
mutations in b at cause loss of function of the transporter system b by defect in folding and trafficking .
in addition , digenic inheritance ( type ab ) has been described . in previous studies ,
these mutations consist of nonsense , missense , splicing , frame shifts , and large sequence rearrangements .
an overview on the most frequent cystinuria mutations in slc3a1 gene in different ethnic groups shows that the selected exons are very important .
considering a few studies on the genetic basis of the cystinuria in the middle east and the population - specific distribution of mutations in the slc3a1 , we tried to find genetic variants in three exons ( 1 , 3 , and 8) of slc3a1 .
twenty - five cystinuria unrelated patients were referred from al - zahra hospital , isfahan university of medical sciences , by a urologist ( 11 women and 14 men ) .
these patients were selected according to the type of cystine stones in the patients who had been subjected to operation for removing kidney stones .
the ethics committee of the medical university of isfahan approved this study according to the national helsinki guidelines ( declaration of helsinki ) ( research project number : 294207 ) .
dna of the samples was extracted according to the standard protocol of kit ( bio genet kit , korea ) .
polymerase chain reaction was used to amplify three pairs of slc3a1 gene primers ( exons 1 , 3 , and 8) in chromosome 2 ( 2p16.3 - 21 ) [ table 1 ] .
primers were designed using primer blast program ( htpp://www.ncbi.nlm.nih.gov / tools / primer - blast ) according to the genomic sequence references available at the genome browser ( gene i d : 6519 , updated on december 6 , 2016 ) .
finally , all samples were sequenced using sanger sequencing method ( macro gene co. korea ) .
according to direct sequencing of exons 1 , 3 , and 8 of slc3a1 gene , we found five patients ( c4 , c6 , c10 , c21 , c23 , and c25 ) who were heterozygote for the polymorphism g38 g in exon 1 [ figure 1a ] .
( c. 114a > c ) makes a synonymous variant of gga ( glycine ) .
the amino acid variant m467k detected in exon 8 in one patient ( c18 ) .
m467k is a missense mutation that changes methionine ( atg ) to lysine ( aag ) at position 179 t / a .
two intronic variants in intron three including c. 610 + 147c > t and c. 610 + 169c > g were also identified in one patient ( c20 ) [ figure 1c and d ] .
( a ) c. 610 + 147c > t , ( b ) c. 610 + 169c > g , ( c ) m467k , ( d ) g38 g
this novel variant is located in exon 1 before initiation codon in 5 untranslated region ( 5utr ) that changes g to a ( c.-29a > g ) .
mutations in the 5utr cause increase or decrease of translation efficiency that recently described as a novel molecular mechanism of disease .
changes in the consensus sequence for the translation initiation may intensify context - dependent leaky scanning of ribosomes and/or initiation from a downstream aug codon .
one previously identified polymorphism in codon 38 ( 114c - a ) was also identified .
g38 g is synonymous variant ( gga change to ggc ) because no amino acid change occurs .
it only changes one codon of amino acid glycine ( gga ) to another codon of glycine ( ggc ) at position 114 of exon 1 ( c. 114a > c ) .
in addition , the frequency of ggc codon in african , american , east asian , european , and south asian populations is 80% , 67% , 38% , 77% , and 86% , respectively . in our study , two known mutations ( c. 610 + 147c > t , c. 610 + 169c > g ) were found in intronic regions of the slc3a1 gene ( intron 3 ) [ table 2 ] .
prevalence of c. 610 + 147c > t intronic mutation is 3% , 1% , 2% , and 1% in african , american , south asian , and european people , respectively .
intronic mutation c. 610 + 169c > g was found in 3% , 1% , 1% 2% of african , american , european , and south asian people , respectively ( http://www.ensemble.org ) .
all patients in our study had percutaneous nephrolithotomy . in our research , we found that1of the 25 ( 4% ) patients had m467k mutation in exon 8 .
in addition , six of patients ( 24% ) showed g38 g polymorphism in exon 1 and 7 out of 25 ( 28% ) had a new mutation in 5utr ( c.-29a > g ) in the slc3a1 gene . both intronic mutations ( c. 610 + 147c > t , c. 610 + 169c > g ) found in 1 out of 25 patients .
in conclusion , our results confirm that cystinuria is a heterogeneous disorder at the molecular level .
this study contributes to the understanding of the distribution and frequency of mutations causing cystinuria in the iranian population .
sm contributed in the acquisition , analysis of data for the work and drafting the work , and agreed for all aspects of the work .
mk contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
ad contributed in the conception of the work , approval of the final version of the manuscript , and agreed for all aspects of the work .
mm contributed in the clinical conception of the work , approval of the final version of the manuscript , and agreed for all aspects of the work .
 background : considering a few studies on the genetic basis of the cystinuria in the middle east and the population - specific distribution of mutations in the slc3a1 , we tried to find genetic variants in three exons ( 1 , 3 , and 8) of slc3a1.materials and methods : in this study , exons 1 , 3 , and 8 of slc3a1 gene of 25 unrelated cystinuria patients searched for genetic variations by polymerase chain reaction and sequencing.results:there were five different variations in our studied population . 
 we found one mutation in the slc3a1 gene including missense variant m467k and identified three polymorphisms : nonsynonymous variant g38 g , c. 610 + 169c > t and c. 610 + 147c > g within the slc3a1 gene , and one new variant.conclusion:our results confirm that cystinuria is a heterogeneous disorder at the molecular level and more studies are needed to identify the distribution and frequency of mutations causing cystinuria in the iranian population . 