| Background and ObjectivesGlucose-6-phosphate dehydrogenase (G6PD) deficiency [MIM305900] is themost common human metabolic disorder in southern China, affecting an estimated400 million people worldwide. Because the G6PD gene is located on the Xchromosome, the clinical symptoms of the disease are usually confined tohemizygous men, although female carriers with a marked expression of the aberrantallele may also suffer. To date, over 150 hematologically important mutationsassociated with different G6PD enzyme abnormalities, including the mutationsresponsible for CNSHA, have been identified at the molecular level from differentethnic populations of the world. Molecular analysis has also revealed that each ethnicpopulation has a characteristic profile of deficient variants. In Southeast Asian, 30different point mutations associated with reduced G6PD enzyme activity have beenidentified in the human G6PD gene. In southern China, 24 different point mutationsassociated with reduced G6PD enzyme activity have been identified in the human G6PD gene. The prevalence of G6PD deficiency in Guangxi Zhuang AutonomousRegion, which has a population of over 49 million, has not been determined. Inaddition, very little is known about the molecular basis of G6PD deficiency in femaleindividuals of southern China because nearly all of data on the incidence rate andmutation spectra was previously observed through the patient-based investigation inmale subjects only. In this report, we investigate the incidence and distribution of thedeficient mutations of the G6PD gene in this region of southern China by usingpopulation-based molecular analysis. The focus of this study will investigate theincidence and distribution of the deficient mutations of the gene and analyze themolecular pathology of females with G6PD deficiency as well as haplotypeassociation of common G6PD deficient mutations.Design and MethodsA total of 4,704 unrelated participants comprising 2,368 males (age 29.45±5.06years) and 2,336 females (age 26.52±3.89 years), whose registered parental origin isfrom 14 different Counties/Districts in Guangxi Zhuang Autonomous Region, wereenrolled for this investigation between October 2002 and October 2003. 98.5％ofthese individuals were ethnic southern Chinese. Among them, most (94.8％) werefrom Guangxi ancestry. Peripheral blood samples were randomly collected from theseparticipants when they received their pre-marriage medical check-up at LiuzhouMunicipal Maternity and Child Healthcare Hospital. For molecular studies of G6PDvariants to clarify their distribution and phenotype within this region, samples werecollected from the two major ethnic population groups, Han and Zhuang whichaccount for 64.3％(3,027/4,704) and 32.5％(1,528/4,704) of the total samples,respectively, were chosen. The rational population-based protocol used in this studyproved to be highly effective in detecting G6PD deficiency. Briefly, two differentscreening procedures, the conventional methemoglobin reduction test followed by themethod advised by the WHO for measurement of G6PD/6PGD ratio, were employed to measure the incidence of G6PD deficiency. The former was primarily used toestimate the prevalence of heterozygote females with G6PD deficiency in this region.In the second, genomic DNA was extracted by standard methods from all suspectedpositive samples and subjected to molecular analysis by a multiplex primerextension/denaturing high performance liquid chromatography (PE/DHPLC) assay,which can simultaneously genotype ten Southeast Asian G6PD deficiency-causingmutations and a silent polymorphism, as previously described. 16 Finally, direct DNAsequencing was performed on all the negative samples as determined by PE/DHPLCin order to detect any novel G6PD gene mutations. In this study, both coding exonsand exon-intron boundaries in the human G6PD gene were completely sequencedusing the ABI Prism DNA Analyzer (Model 377). As a control for this study, werandomly selected 129 samples from normal individuals and determined the G6PDmutation status by PE/DHPLC. Of these 129 samples, we sequenced all 65 femalesamples except for the two samples with known mutations detected by PE/DHPLC.The criteria used to make diagnosis of G6PD-deficiency in this study included: (ⅰ) apositive enzyme test by both MRT and G6PD/6PGD ratio assay or (ⅱ) a positivemutation as determined by molecular analysis using PE/DHPLC or DNA sequencing.Blood counts and red-cell indices were assessed in all the samples using standardprocedures.Results and InterpretationTotal numbers of G6PD-deficient chromosomes in males were 176, with a highfrequency of 7.43％for this enzyme abnormality in Guangxi population. A total of341 chromosomes with different known mutations were characterized in our study.We observed a total of 27 different genotypes from 320 G6PD-deficient individualsans three apparently normal individuals characterized through molecular analysis in this study, including eight ones in 161 hemizygotes, twelve ones in 135 heterozygotes,and six ones in 21 homozygotes, respectively. In addition, 15 males and 26 femaleshad unknown mutations. Comparison of the mutation frequency results of the ratesbetween Han with 16.38％(249/1,520) and Zhuang with 15.39％(117/760)demonstrating that there were no significant statistical difference(X2=2.368, P＞0.05)between ethnic backgrounds. The mutation profile from Guangxi ZhuangAutonomous Region showed that a battery of 4-6 common mutations accounted forabout 85％of these mutations. The distribution of the five dominant mutantalleles(1388G＞A, 1376G＞T, 95A＞G, 1024C＞T and 871G＞A) between Han andZhuang people was not statistically different for each of the five groups (X2=0.620, P＞0.05). We then compared two phenotypes from female heterozygotes with thesame genotypes that were isolated from the group of 39 detected individuals and thegroup of 108 detected individuals by PE/DHPLC genotyping. The results showed asignificant difference in phenotypic features of G6PD/6PGD measurements betweenthe two groups and are compatible with the rule for incomplete inheritance of thisX-linked disorder in females. This suggests that they may have variable enzymeactivities even though it was the same mutation in female heterozygotes. Statisticanalysis showed that there were significant enzyme activity-related differences inboth the percentages of MetHb and the G6PG/6PGD ratio between heterozygote andhemizyote (P=0.000, P＜0.05), and heterozygote and homozygote (P=0.000, P＜0.05).There was no statistically significant difference between hemizyote and homozygote(MetHb％: P=0.272, P＞0.05; G6PD/6PGD: P=0.310, P＞0.05), in addition to asignificant difference existed between the normal control group and theG6PD-deficient group (P=0.000, P＜0.05). The mutation profile of 15 deficient allelesidentified in this region showed that about 85％of them was accounted for by abattery of five common mutations involved in G6PD Kaiping, G6PD Canton, G6PD Gaohe, Chinese-5, and G6PD Viangchan; approximately 4％accounted for by theremaining 10 rare mutations involved in Chinese-4, G6PD Liuzhou, G6PD Fushan,G6PD Songklanagarind, G6PD Asali, G6PD Hechi, G6PD Miaoli, G6PD Nanning,G6PD Laibin, and G6PD Shunde; nearly 14％accounted for by uncharacterizedvariant. Among these variants, four novel mutations in G6PD gene were G6PDLiuzhou442A producing a Glu 148 to Lys change, G6PD Nanning703T results in a Leu235 to Phe substitution, G6PD Laibin1414C results in an Ile 472 to Leu substitution,and a double mutations responsible for G6PD Hechi202A/871A. Additionally, two raremutations, G6PD Songklanagarind and G6PD Asali known to be present in Japan orThailand, were firstly identified in Chinese patients. The data on haplotyping 176male and 6 female deficient chromosomes showed that the haplotype (--+--) isdominant, covering over 94％of the haplotype patterns in Chinese G6PD-deficientindividuals, supporting the hypothesis that mutations occurring at nt 95, 1024, 1376,and 1388 could be derived from this ancient common haplotype. The two mutations,all 871G＞A mutations and very few 1376G＞T mutations, are linked with a minorhaplotype (--+++), indicating that the mutation happened more recently.Additional analysis of newly identified deficient alleles in six females (three femaleswith G6PD Liuzhou442A, one with G6PD Laibin1414C, one with G6PD Nanning703T andone with G6PD Songklanagarind) displayed the same haplotype (--+--), furthersuggesting that they may have arisen from a common ancestral origin. The frequencyof the common polymorphism 1311T in males is 7.8％, which is equivalent to thetesting rate of 1311T in cis with intron 11-93 C. Among the female heterozygotes, wefound a group of the trans-compound heterozygotes of 1311T with the commonG6PD-deficient mutations except for three which were the 1311 T in cis with 871G＞A (G6PD Viangchan) mutation. All of the 8 male patients with a combination of871 G＞A responsible for the variant G6PD Viangchan and the 1311 C＞T was found to be the cis-compound heterozygote which is similar to the linkage disequilibrium(LD) between mutation 871A and polymorphic site 1311 T previously observed in theAsian population.Overall, the rational population-based protocol used in this study proved to behighly effective in detecting G6PD deficiency. It could be used to extend theknowledge of molecular defects of G6PD gene in other geographical regions of Chinaand also in other countries. This protocol could be used for investigate the prevalenceof G6PD deficient gene in different geographical areas. From the data obtained in ourstudy, the high frequency of the G6PD deficiency and the elucidation of mutationpatterns would enhance the basic knowledge of this disorder and also develop a morerational approach to population screening for prevention and counseling of disease inthis area.
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