glucose-6-phosphate dehydrogenase ( g6pd ) deficiency is one of the most common hereditary hemolytic disorders in human , affecting around 400 million people worldwide .
the majority of deficient individuals live in tropical and subtropical regions where malaria is still endemic or has been eradicated only recently .
in fact , g6pd deficient individuals have been protected from severe forms of malaria in these areas .
although most of individuals with g6pd deficiency are usually asymptomatic , deficient subjects may suffer from neonatal jaundice , severe non - spherocytic hemolytic anemia or acute hemolytic anemia induced by infection , ingestion of fava beans or some drugs .
the severity of these serious consequences of g6pd varies based on different gene mutations which cause different levels of residual enzyme activity . therefore to prevent the above complications , it is important to investigate their molecular bases .
this gene is located on the xq28 region with a length of 18.5 kb , which contains 13 exons and 12 introns . since g6pd deficiency is an x - linked recessive disorder , it is more frequent in males than females .
glucose-6-phosphate dehydrogenase enzyme , the product of g6pd gene , catalyzes the first step of the pentose phosphate pathway ( ppp ) , which provides cells with pentoses and reduction power in the form of nicotinamide adenine dinucleotide phosphate ( nadph ) .
nicotinamide adenine dinucleotide phosphate cofactor is required for various redox reactions , and protects cells against oxidative stress via glutathione and catalase .
glucose-6-phosphate dehydrogenase is the only source of nadph in erythrocytes , so any oxidative stress in g6pd deficient red blood cells may results in hemolytic anemia . approximately 140 mutations and 400 biochemical variants have been reported for this enzyme till now .
the g6pd cosenza mutation was described for the first time in the of , southern .
previous investigations have revealed that g6pd cosenza ( g1376c ) , which is a common g6pd mutation in some parts of iran , has a variable frequency ranging from 0% to 12.33% .
given the variability and high frequency of g6pd cosenza in iran , in the present study we have characterized g6pd cosenza among deficient individuals in the province of khuzestan , which is located in the southwest of the country bordering iraq and the persian gulf with a population of about five million mostly iranian arabs .
screening study was performed on 1064 randomly selected blood samples from volunteer male donors referring to ahvaz blood transfusion center from february to april 2008 .
screening test for the diagnosis of g6pd deficiency was done by fluorescent spot method ( sigma ) .
eighty - one ( 7.6% ) of them were found to be severely g6pd deficient .
however blood sample were taken only from 79 deficient male blood donors for next studies . in order to identify g6pd molecular characterization ,
231 g6pd deficient blood samples were collected from 79 screened male blood donors and 152 individuals ( 116 males and 36 females ) who were referred to hospitals of khuzestan province with a history of favism , acute anemia or neonatal jaundice .
all samples were collected into 0.5 m edta ( becton dickinson , ) solution , carried on ice and kept at -70 c for dna extraction . because g6pd mediterranean ( c563 t ) is reported as the most common mutation in middle east and some provinces of iran , at first we analyzed all samples for this mutation .
finally 64 ( 55 males and 9 females ) out of 231 samples were recognized without mediterranean mutation , which were then analyzed to identify cosenza mutation.genomic dna was extracted from leukocytes by using " picopure  " dna extraction kit from molecular devices ( san diego , ca ) .
mutation site is located in exon 12 of g6pd gene . for detection of the cosenza mutation ,
exon 11 - 13 of g6pd gene was selectively amplified by pcr method using f - cos ( 5-gca gcc agt ggc atc agc aag-3  ) and r - cos ( 5-ggg aag gag ggt ggc cgt gg-3  ) primers .
polymerase chain reaction ( pcr ) assay was performed in final volume of 25 l .
pcr reaction mix contained 10x pcr buffer , 10 mm of each deoxynucleotide triphosphate , 25 pmol of each primer , 0.5 g genomic dna , 2 u / ml of taq dna polymerase and 50 mm mgcl2 .
the pcr reaction was carried out for 30 cycles as follows : 10 cycles ( 94 c for 30 seconds , 68 c for 1 min and 72 c for 30 seconds ) and 20 cycles ( 95 c , 65 c , 72 c each temperature for 1 min ) . in order to certify the fidelity of pcr , amplified segments
since the mutation creates an eco81i recognition site ( figure 2 ) , this endonuclease was used to perform restriction fragment length polymorphism ( rflp ) analysis .
cozenza pcr products were digested by eco81i enzyme ( fermentas gmbh , ) at 37 c , overnight .
among the 231 g6pd deficient individuals ( a total of 267 alleles ) , 195 ( 84.1% ) were males and 36 were females .
only 64 samples ( 55 males and 9 females ) out of 231 deficient subjects did not have g6pd mediterranean .
cosenza pcr product was a 548 bp fragment , which appeared as two fragments with 232 bp and 316 bp lengths after digestion by eco81i on 2% agarose gel in mutant subjects ( figure 3 ) .
therefore the mutation relative rate and allele frequency in khuzestanian deficient subjects are 2.6% and 0.023 , respectively .
polymerase chain reaction ( pcr ) products related to glucose-6-phosphate dehydrogenase cosenza mutation on 1.5 % agarose gel .
lane 1 : size marker 1 kbp , lane 2 : negative control , lanes 3 , 4 , 5 , 6 , 7 , 8 and 9 : cosenza pcr products with 548 bp length oligonucleotide primers f - cos and r - cos amplify a 548pb fragment across exon 11 and 13 of the glucose-6-phosphate dehydrogenase gene that after digestion by eco81i appeared as two fragments with 232 bp and 316 bp restriction digestion analysis of polymerase chain reaction ( pcr ) products related to glucose-6-phosphate dehydrogenase cosenza mutation with eco81i enzyme on 2% agarose gel .
lane 1 : size marker 100 bp , lane 2 : pcr product , lanes 3 , 4 , 5 , 6 , 7 , 8 and 9 : samples without cosenza mutation , lane 10 : hemizygote male with cosenza mutation
g6pd deficiency is a very prevalent disorder in africa , southern europe , south east asia , oceania and middle east especially neighboring persian gulf countries including iran , kuwait , united arab emirates , iraq , bahrain , and oman , with a prevalence of 11.55 , 5.51 , 8.7 , 6.1 , 26.45 and 26 - 29% , respectively .
khuzestan province is located in the south west iran , bordering iraq and the persian gulf .
the prevalence of g6pd deficiency among male khuzestanian blood donors was reported to be 7.6% .
this prevalence is obviously higher than the 6 % reported earlier for male blood donors in fars province of southern iran .
however , one other recent study showed that the overall prevalence of g6pd deficiency among male and female children in the city of shiraz ( fars province ) was 11% .
since g6pd deficiency is so frequent in khuzestan , it is very important and desirable to fully identify the molecular basis of this disorder .
our previous study revealed that g6pd mediterranean ( c563 t , ser188phe ) was the most common mutation in khuzestan , like the other provinces of iran . to pursue our investigation
cosenza mutation , which was initially described in the north of calbaria , southern italy , by frequency of 1.9% , is caused by 1376 g  c ( 459 arg  pro ) substitution .
its phenotype is associated with a severe enzyme deficiency ( enzyme activity less than10 % ) .
thus far , g6pd cosenza has been identified in some parts of italy , daltamation region of south croatia , and some parts of iran including mazandaran , kermanshah , and hormozgan .
however , it hasnt been found in gilan , golestan , sistan and balochestan , khorasan , and fars provinces . the highest incidence ( 37.5% ) of g6pd cosenza
it not known whether this distribution of g6pd cosenza is the result of a common ancestry or an independent origin in mediterranean basin and middle east .
we have detected cosenza mutation in 6 of 231 samples , resulting in a prevalence of 2.6% and allele frequency of 0.023 .
therefore the incidence of mutation in khuzestan is higher than that in , but lower than those in hormozgan and mazandaran .
kermanshah , hormozgan and mazandaran are provinces that are respectively located in the western , southern and northern parts of ( table 1 ) .
the great difference between the incidences of g6pd cosenza in some parts of could be explained by immigration issues , which might have induced a flow of gene from countries . or
alternatively , it might be due to the origin of ethnic groups , which may be clarified by studying additional markers in populations .
the variation in the prevalence on cosenza mutation may be partially comparable to that of thalassemia in the south of the country .
we also studied the association of mediterranean and cosenza mutation with ( 1311ct ) haplotype .
the haplotype analysis revealed that unlike mediterranean g6pd , mutation was associated with ( 1311c ) haplotype .
fifty eight samples , which have not mediterranean and mutations , are kept for identification of other g6pd mutations .
the findings of the present study indicate that g6pd cosenza is a common mutation in khuzestanian g6pd deficient individuals .
 glucose-6-phosphate dehydrogenase ( g6pd ) deficiency is one of the most common hereditary enzymatic disorders in human , increases the vulnerability of erythrocytes to oxidative stress . 
 it is also characterized by remarkable molecular and biochemical heterogeneity . 
 according to previous investigations , g6pd cosenza ( g1376c ) is a common g6pd mutation in some parts of . 
 therefore in the present study we have characterized mutation among g6pd deficient individuals in khuzestan province . in order to identify g6pd cosenza , we analyzed the g6pd gene in 64 samples out of 231 deficient individuals who had not g6pd mediterranean mutation , using pcr- restriction fragment length polymorphism ( rflp ) method . 
 g6pd cosenza mutation was found in 6 males of 231 samples , resulting in the relative rate of 2.6% and allele frequency of 0.023 among khuzestanian g6pd deficient subjects . 
 a comparison of these results with previous findings in some parts of suggests that g6pd cosenza is a common mutation in khuzestanian g6pd deficient individuals . 
Introduction
Patients and Methods
Results
Discussion
Conclusion
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