Diverse point mutations result in glucose-6-phosphate dehydrogenase (G6PD) polymorphism in Taiwan.

Glucose-6-PHOSPHATE dehydrogenase (G6PD; EC 1.1.1.49) deficiency is the most common human enzymopathy, affecting more than 200 million people worldwide. Although greater than 400 variants have been described based on clinical and biochemical criteria, little is known about the molecular basis of these G6PD deficiencies. Recently, the gene that encodes human G6PD has been cloned and sequenced, which enables us to examine directly the heterogeneity of G6PD at the DNA level. During the past 10 years, we examined the G6PD activity in 21,271 newborn Chinese infants (11,400 males and 9,871 females) and identified 314 (2.8%) males and 246 (2.5%) females having low G6PD activity. The G6PD gene from 10 randomly selected affected individuals and their relatives was polymerase chain reaction (PCR) amplified, subcloned, and sequenced. Our results indicate that at least four types of mutation are responsible for the G6PD polymorphism in Taiwan. The first type of mutation (487 G----A) was found in an affected Chinese with a G to A change at nucleotide 487, which results in a (163)Gly to Ser substitution. The second type of mutation (493 A----G) is a novel mutation that has not been reported in any other ethnic group and was identified in two affected Chinese. This mutation causes an A to G change at nucleotide position 493, producing an (165)Asn to Asp substitution. Interestingly, the 487 G----A and 493 A----G mutations create Alu I and Ava II recognition sites, respectively, which enabled us to rapidly detect these two mutations by PCR/restriction enzyme (RE) digestion method. The third mutation (1376 G----T) was found in four affected Chinese. This mutation causes a G to T change at nucleotide position 1376 that results in an (459)Arg to Leu substitution. The 1376 G----T mutation seems to be the dominant allele that causes G6PD deficiency in Taiwan. Finally, two affected Chinese were identified as having the fourth mutation (1388 G----A). This mutation causes a G to A change at nucleotide 1388 that produces an (463)Arg to His substitution. Our studies provide the direct proof of the genetic heterogeneity of G6PD deficiency in the Chinese populations of Taiwan and the PCR/RE digestion method is suitable for simultaneous detection of the 487 G----A and 493 A----G mutations.     

Molecular characterisation of red cell glucose-6-phosphate dehydrogenase deficiency in Singapore Chinese

Sixty-two G6PD deficient Chinese males have been investigated for the presence of seven mutations of the coding region of the G6PD gene by natural and artifically created amplified restriction sites. The results show that the G to T substitution at nucleotide (nt) 1376 and G to A substitution at nt 1388 represent 24% and 21% of G6PD deficiency, respectively, in the Singapore Chinese; 37% of the sample could not be characterised. The remaining samples were identified as follows: 10% C→T at nt 563, 5% A→G at nt 95, and 3% C→T at nt 1024. The G to A substitution (nt 487) and the substitution A→G (nt 493) were not present in this sample. None of the subjects with the Mediterranean mutation (563 C→T) had the silent mutation at 1311 (C→T). This study confirms the extreme molecular heterogeneity of the G6PD gene in the Chinese. © 1994 Wiley-Liss, Inc.     

Neonatal jaundice and molecular mutations in glucose-6-phosphate dehydrogenase deficient newborn infants

Molecular mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene and clinical manifestations of neonatal jaundice in 112 male and 50 female Chinese neonates with G6PD deficiency were studied. In the 112 males, the nucleotide (nt) 1376 (G→T) mutation was the dominant type (50.0%), followed by nt 1388 (G→A) (16.1%), nt 493 (A→G) (8.0%), nt 1024 (C→T) (6.2%), nt 95 (A→G) (5.4%), nt 392 (G→T) (1.8%), nt 487 (G→A) (1.8%), nt 871 (G→A) (0.9%), and nt 1360 (C→T) (0.9%). The nt 871 variant has not been reported in Taiwan before. The occurrence rates for nt 1376, nt 1388, nt 493, nt 95, and nt 1024 mutations in the 50 females were 44.0%, 18.0%, 12.0%, 6.0%, and 6.0%, respectively. The type of G6PD mutation in 10 male and 7 female neonates has not been identified yet. Although G6PD deficient neonates had higher frequency of phototherapy than G6PD normal neonates in both sexes, a significant difference in the prevalence of hyperbilirubinemia (peak bilirubin ≥ 15.0 mg/dl) between G6PD deficient and normal neonates was found only in males. Further analysis showed that duration of phototherapy was longer in G6PD deficient male neonates than in the control group, while the outcome of phototherapy was better in subjects with non-nt 1376 mutations than subjects with the nt 1376 mutation. Most (78.3%) of the 23 G6PD deficient neonates who subsequently suffered from neonatal hyperbilirubinemia carried the nt 1376 mutation. The results of this study indicate that the nucleotide substitution at 1376 is the most common and important mutation for G6PD deficiency in Chinese neonates in Taiwan. © 1996 Wiley-Liss, Inc.     

Diagnosis of glucose-6-phosphate dehydrogenase (G6PD) mutations by DNA amplification and allele-specific oligonucleotide probes.

We have recently identified that at least four types of mutation are responsible for the glucose-6-phosphate dehydrogenase (G6PD) polymorphism in the Chinese of Taiwan. Two mutations (487 G-->A and 493 A-->G) occurring at nucleotide position 487 and 493, respectively, create Alu I and Ava II recognition sites which enabled us to directly examine these two mutations by PCR/restriction enzyme (RE) digestion. However, the other two mutations (1376 G-->T and 1388 G-->A), which do not generate any recognizable restriction sites, were detected by DNA sequencing method which is not suitable for rapid diagnosis. Using the PCR technique, we have successfully developed a simple and rapid method for the detection of 1376 and 1388 mutations. This method involves the selective amplification of a DNA fragment from human G6PD gene with specific oligonucleotide primers, followed by hybridization with allele-specific oligonucleotide (ASO) probes. Using the PCR/ASO and PCR/RE methods, we have successfully examined two families and 20 unrelated subjects with G6PD deficiency. Our results indicate that the PCR/ASO method is suitable for the rapid determination of 1376 and 1388 mutations. The combined use of PCR/ASO and PCR/RE methods will be suitable for rapid diagnosis of four known G6PD mutations in Chinese.     

Rapid detection of six common Chinese G6PD mutations by MALDI-TOF MS

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked hereditary enzymopathy. We describe here the techniques based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and multiprimer extension (multi-PEX) to detect the most common Chinese G6PD mutations, which are the single-point mutations G-->T at nt 1376, G-->A at nt 1388, A-->G at nt 95, G-->T at nt 392, C-->T at nt 1024, and C-->T at nt 1311. Fifteen samples were genotyped using this method coupled with direct sequencing, after identification of G6PD mutations by ARMS. In this study, we identified a mutation G-->T at nt 1376, which had been G-->A at nt 1388 using ARMS, while the result of sequencing corresponds with ours. This indicates the reliability of this method. Furthermore, since it can scan six common Chinese G6PD mutations simultaneously in one mass spectrum, this approach could be used to fast diagnose these G6PD mutations accurately in large-scale analysis.     

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Chinese infants with or without severe neonatal hyperbilirubinaemia

Summary. To characterize mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene in Chinese infants, we studied 213 G6PD-deficient infants without blood exchange transfusion (BET) therapy, and 34 patients who required BET therapy for their severe hyperbilirubinaemia after birth. Nine different point mutations were characterized in all infants. Of these mutations, the G to T substitution at cDNA nucleotide (nt) 1376, which accounts for the mutations in 131 (53.0%) neonates, followed by G to A substitution at nt 1388 in 18 (10.5%) infants, A to G substitution at nt 493 in 17 (6.9%) infants, A to G substitution at nt 95 in 10 (4.1%) infants, C to T substitution at nt 1024 in six (2.4%) infants, and G to T substitution at nt 392 in three (1.2%) infants, G to A substitution at nt 487 in two (0.8%) infants, C to T substitution at nt 1360 in two (0.8%) infants and C to T substitution at nt 592 in two (0.8%) infants. Mutations in 48 (19.5%) G6PD-deficient infants were not characterized. Most (64.7%) mutations in the G6PD-deficient infants who required BET therapy after birth result from a G to T substitution at nt 1376. The enzyme activity of G6PD deficient infants who required BET therapy is significantly lower than for those who did not, even in a group with the same variant (as in 1376 mutation). Severe neonatal jaundice requiring BET therapy can take place with the majority of variants encountered in this area.     

Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients of Chinese descent and identification of new base substitutions in the human G6PD gene

The underlying DNA changes associated with glucose-6-phosphate dehydrogenase (G6PD)-deficient Asians have not been extensively investigated. To fill this gap, we sequenced the G6PD gene of 43 G6PD- deficient Chinese whose G6PD was well characterized biochemically. DNA samples were obtained from peripheral blood of these individuals for sequencing using a direct polymerase chain reaction (PCR) sequencing procedure. From these 43 samples, we have identified five different types of nucleotide substitutions in the G6PD gene: at cDNA 1388 from G to A (Arg to His); at cDNA 1376 from G to T (Arg to Leu); at cDNA 1024 from C to T (Leu to Phe); at cDNA 392 from G to T (Gly to Val); at cDNA 95 from A to G (His to Arg). These five nucleotide substitutions account for over 83% of our 43 G6PD-deficient samples and these substitutions have not been reported in non-Asians. The substitutions found at cDNA 392 and cDNA 1024 are new findings. The substitutions at cDNA 1376 and 1388 account for over 50% of the 43 samples examined indicating a high prevalence of these two alleles among G6PD-deficient Chinese. Our findings add support to the notion that diverse point mutations may account largely for much of the phenotypic heterogeneity of G6PD deficiency.     

Neonatal screening for glucose-6-phosphate dehydrogenase deficiency in Taiwan

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathic disease in Taiwan. The mass neonatal screening of G6PD deficiency by fluorometric spot test in Taiwan was started with a pilot program in 1984. The nationwide screening was started on July 1, 1987, and a follow-up system comprising of eighteen referral hospitals, including outlying islands, was organized for confirmatory test, medical care and genetic counseling. From July 1987 to December 1997, 2,971,192 heel blood samples collected on filter paper from 1,143 delivery units were screened by four neonatal screening centers. 46,570 cases were confirmed as G6PD deficiency is estimated to be around 2.1% (male 3.1%, female 0.9%) in Taiwan. The coverage rate of neonatal screening was 99% in 1997. To assess the reliability of the confirmatory test, an external quality assurance (QA) program for G6PD assay was developed. Periodically, 3 or 5 lyophilized quality control materials with different activities of G6PD were sent to each referral hospital by speed post delivery in dry ice. From January 1988 to June 1998, 85 QA services were performed. Two hundred and seven (13.5%) abnormal QA results were found, which were attributed to clerk (11.6%), procedural (16.4%), and instrumental errors (47.3%). In aid to confirm G6PD deficiency, a method to detect the G6PD mutation by using the dried blood samples was developed. The frequencies of the mutant alleles in Taiwan were determined to be 46.8% (1376G > T), 16.2% (1388G > A), 7.9% (95A > G), 6.5% (493A > G), 5.6% (392G >T), 4.6% (1024C > T), 0.5% (487G > A) and 0.5% (519C > G), respectively.     

贵州省从江县侗族人群葡萄糖-6-磷酸脱氢酶基因突变型的检测

目的了解贵州省从江县侗族葡萄糖-6-磷酸脱氢酶(Glucose-6-phosphate dehydrogenase,G6PD) 缺乏症的发生率、基因突变类型及特点。方法对贵州省从江县侗族524人采用四氮唑蓝定性法进行G6PD缺乏症初筛、G6PD／6PGD比值法验证．再经自然引物及错配引物介导的聚合酶链反应／限制性酶切分析法检测中国人常见的9种基因突变型,对于未定型采用变性梯度凝胶电泳法(DGGE)检查外显子2、8、9、12基因突变情况。结果 G6PD缺乏症34例,检出率为6．49％,其中检出G1388A突变4例、C592T突变18例。未定型12例经DGGE检测外显子突变情况,未发现突变,有待于进一步对其余外显子进行研究。结论贵州省从江侗族是G6PD缺乏症的高发区。592 C→T突变型为该地该民族常见突变型,而不是中国人常见的G1376T、G1388A或A95G突变型。此次基因突变型调查为了解贵州省少数民族G6PD缺乏症的分布特征提供了原始数据。     

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in Yogyakarta and its surrounding areas

Glucose-6-phosphate dehydrogenase (G6PD) is one of the enzymes needed by the erythrocyte to generate ATP from ADP. Deficiency of this enzyme can lead to hemolysis of red blood cells. Being a malaria endemic area, Indonesia possibly has a high incidence of G6PD deficiency. It is estimated that 2-6% of the population are carriers. In 1996, we detected 145 neonates with G6PD deficiency using the formazan ring method. Among the males, 6.2% had moderate and 1.4% had low enzyme activity; females had enzyme activity in the normal range. Using the Sigma kit, Tashimi et al in 1995 examined 111 neonates in Yogyakarta, none of which was identified as "deficient". There was no correlation between erythrocyte hemolysis and G6PD enzyme content. Interestingly, using the same Sigma kit. Soro et al in 1994 found that among 134 individuals of Batak descent, 10 males (43.48%) and 9 females (8.11%) were G6PD deficient. These were similar to the results reported by Pramuji et al in 1995 for the people around Palembang. Since the G6PD gene is located on the X chromosome, this is a peculiar result thus further studies need to be done. In cooperation with Harvard University, Sumantri et al in 1995 described 14% as carriers. Molecular analysis among these 16 Javanese males showed the following mutations--nt563 (C->T) in 5 cases, nt1376 (G->T) in 3 cases, nt487 (G->A) in 2 cases, nt1311 (C->T) in 1 case with the remaining variants unknown.     

Definition of the mutations of G6PD Wayne, G6PD Viangchan, G6PD Jammu, and G6PD 'LeJeune'.

We report the nucleotide (nt) substitutions of four unrelated glucose-6-phosphate dehydrogenase (G6PD)-deficient males. Only the mutation of G6PD Wayne was unique. It was a nt 769 C----G substitution causing a deduced substitution of glycine for arginine at amino acid 257. This mutation is in a region in which G6PD mutations have previously been associated with chronic hemolytic anemia. The mutation of G6PD Jammu and G6PD Viangchan were identical: a G----A mutation at nucleotide 871, predicting a Val----Met substitution at amino acid 291. However, these two variants differ with respect to the 1311 polymorphism, suggesting that they may have arisen independently. Enzyme from a child with chronic hemolytic anemia, designated G6PD 'LeJeune', proved to be due to a G----T substitution at nt 637, a change identical with that in 3 unrelated patients who had been reported previously as having G6PD Gastonia, Minnesota and Marion. These findings support the suggestion that both polymorphic and sporadic G6PD deficiency mutations in unrelated persons with G6PD deficiency are often the same, even when thought to be distinct on the basis of biochemical characterization.     

Multiple G6PD mutations are associated with a clinical and biochemical phenotype similar to that of G6PD Mediterranean

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common red cell abnormalities, is characterized by a wide clinical, biochemical, and molecular heterogeneity. In this study we have determined the molecular basis of G6PD deficiency in a sample of 70 male subjects, originating from different parts of Italy, who all shared a clinical and biochemical phenotype identical or very similar to that of G6PD Mediterranean, the most common variant in Italy. In 59 cases (84%) we found the mutation 563 C --> T, previously known to be underlying the G6PD Mediterranean and the two polymorphic variants G6PD Cagliari and G6PD Sassari. From the remaining 11 we amplified the entire coding region of G6PD in 8 different fragments and subjected them to nonradioactive single-strand conformation analysis. Direct sequencing was then performed on abnormal fragments. By this approach we found six cases (8.5%) with 1360 G --> A mutation (G6PD Union) and two cases (2.8%) with 1376 G --> C (G6PD Cosenza). In the remaining three samples we found two other mutations: 1342 A --> G (two cases, 2.8%) and 1052 G --> T (one case, 1.4%). These two molecular defects have never been described before and were designated by us as G6PD S. Antioco and G6PD Partenope, respectively. Haplotype analysis suggested that all the non-Mediterranean mutations occurred independently on a normal G6PD allele. This study shows that the G6PD Union mutation has a high polymorphic frequency in the Italian population and that the genetic heterogeneity of G6PD Mediterranean-like variants is higher at the molecular level than expected from biochemical characterization.     

Molecular characterization of G6PD deficiency in Cyprus

In the present study, we determined the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Cyprus using two different procedures in two separate adult population groups: a semiquantitative fluorescence test on blood spotted on filter paper and a quantitative spectrophotometric test on liquid blood. The frequency of G6PD deficiency among healthy adult males was found to be 5.1% using the semiquantitative procedure and 6.4% using the quantitative procedure. Neither method was able to detect all the expected female heterozygotes (5.3% and 47.1% of the expected number, respectively). A total of 21 male hemizygotes, 1 female homozygote and 9 female heterozygotes that tested positive for G6PD deficiency were studied at the molecular level. All 32 chromosomes were genotyped and five different mutations were identified. The Mediterranean mutation in exon 6 (563C-->T) (Ser188Phe) was found to be the most common variant in the Cypriot population, accounting for 52.6% of the deficient alleles. In the remaining chromosomes, four different mutations were identified: three known mutations, Kaiping 1388G-->A (Arg463His), Chatham 1003G-->A (Ala335Thr) and Acrokorinthos 463C-->G (His155Asp), and one previously undescribed mutation in exon 3, 148C-->T (Pro50Ser), which we called G6PD Kambos. We conclude that the frequency of G6PD deficiency in Cypriot males is 6.4%, and that this deficiency is the result of several different mutations. Although all the individuals carrying the Mediterranean variant can be detected using a semiquantitative screening method, a quantitative enzyme measurement is required to detect the G6PD variants with less severe enzyme deficiencies, while the most appropriate method for heterozygote detection is DNA analysis.     

Molecular Characterization of G6PD Deficient Variants in Nineveh Province,Northwestern Iraq

Glucose-6-phosphate dehydrogenase (G6PD) deficiency considered to be the commonest inherited enzymopathies disorders worldwide including Iraq. Studies have addressed its prevalence and molecular characterization in several parts of the country, but no data were available from Nineveh province, northwestern-Iraq regarding molecular basis of this inherited enzymopathy. To determine the molecular basis of G6PD deficient variants in Nineveh province. A total of 61 G6PD deficient male individuals from Nineveh province were enrolled in this study. DNA from all enrolled individuals were extracted and analyzed for four deficient molecular variants using a polymerase chain reaction–restriction fragment polymorphism method. These deficient variants were G6PD-Mediterranean (563 C→T), G6PD-Chatham (1003 G→A), G6PD-A-(202 G→A) and G6PD-Cosenza (1376 G→C). Also enrolled individuals were screened for silent 1311 (C→T) mutation. It was found that 46 (75.41 %) were G6PD-Mediterranean, 1(1.64 %) were G6PD-Chatham, another 1(1.64 %) were G6PD-A-, and 13 (21.31 %) were remained uncharacterized. Also all G6PD-Mediterranean as well as one uncharacterized individuals were carriers of silent 1311 (C→T) mutation. This study documented that G6PD-Mediterranean constitute the bulk of G6PD deficient variants in this province and G6PD-Chatham and A- were encountered less frequently. Also that silent 1311 (C→T) mutation were common among G6PD-Mediterranean deficient variants individuals.     

G6PD Viangchan and G6PD Mediterranean are the main variants in G6PD deficiency in the Malay population of Malaysia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked red blood cell enzymopathy common in malaria endemic areas. Individuals affected by this disease show a wide variety of clinical signs of acute hemolytic anemia. Mutations of the G6PD gene in the Malay population with G6PD deficiency in Kelantan, a state in North East Malaysia were studied. Ninety-three individuals with G6PD deficiency were subjected to mutation analysis of the G6PD gene using polymerase chain reaction based techniques of multiplex PCR. Of the ninety-three DNA samples studied, molecular defects were identified in 80 cases (86%). Variants were heterogeneous - 28.7% were found to have a G to A nucleotide change at nucleotide 871 of the G6PD gene (G871A), corresponding to G6PD Viangchan. The other major mutations were G6PD Mediterranean, G6PD Vanua Lava, G6PD Coimbra, G6PD Kaiping, G6PD Orissa, G6PD Mahidol, G6PD Canton and G6PD Chatham. These results showed that there are heterogeneous mutations of the G6PD gene associated with G6PD deficiency and that G6PD Viangchan and G6PD Mediterranean account for the main variants in G6PD deficiency among the Malay population in Malaysia.     

贵州省荔波县瑶族葡萄糖-6-磷酸脱氢酶缺陷症基因突变研究

目的了解贵州省荔波县瑶族葡萄糖-6-磷酸脱氢酶(G6PD)缺陷症的发生率、基因突变类型及特点。方法对贵州省荔波县瑶族586人采用四氮唑蓝定性法进行G6PD缺陷症初筛、G6PD／6PGD比值法验证,再经自然引物及错配引物介导的聚合酶链反应／限制性酶切分析法检测中国人常见的9种基因突变型。结果筛出G6PD缺陷阳性样本45例,基因频率为7．68％,其中检出G1388A突变15例、G1376T突变7例。结论贵州省荔波县瑶族是G6PD缺陷症的高发区,该地该民族常见突变型是中国人常见的G1376T、G1388A突变型。本调查为了解贵州省少数民族G6PD缺陷症的分布特征提供了原始数据。     

Hemolytic crisis after excessive ingestion of fava beans in a male infant with G6PD Canton

After ingesting fava beans, a 26-month-old Chinese-Japanese male infant showed a sickly complexion and yellowish-brownish skin and was hospitalized. Severe hemolytic anemia was observed on admission, and transfusion of 200 ml of packed red cells was required. Red cell enzyme assay revealed that the patient and the mother were deficient in glucose-6-phosphate dehydrogenase (G6PD). Subsequent molecular analysis showed that the patient had a missense mutation 1376 G to T (G6PD Canton) and his mother was a homozygote for the mutation. The patient was a son of a Chinese (Taiwanese) mother and a Japanese father. Although G6PD deficiency is rare in the original Japanese population, the number of "imported" cases could be rising rapidly. This is the first reported Japanese case of G6PD deficiency with G6PD Canton.     

Chronic nonspherocytic hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency: report of two families with novel mutations causing G6PD Bangkok and G6PD Bangkok Noi

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common hereditary enzymopathies worldwide. Mostly G6PD deficient cases are asymptomatic though they may have the risk of neonatal jaundice (NNJ) and acute intravascular hemolysis during oxidative stress. Chronic nonspherocytic hemolytic anemia (CNSHA) due to G6PD deficiency is rare. In Thailand, one case was reported 40 years ago and by biochemical study this G6PD was reported to be a new variant G6PD Bangkok. We, herein, report two families with CNSHA due to G6PD deficiency. In the first family, we have been following up the clinical course of the patient with G6PD Bangkok. In addition to chronic hemolysis, he had three acute hemolytic episodes requiring blood transfusions during childhood period. Multiple gallstones were detected at the age of 27. His two daughters who inherited G6PD Bangkok from him and G6PD Vanua Lava from his wife are asymptomatic. Both of them had NNJ and persistent evidences of compensated hemolysis. Molecular analysis revealed a novel missense mutation 825 G→C predicting 275 Lys→Asn causing G6PD Bangkok. In the second family, two male siblings are affected. They had NNJ and several hemolytic episodes which required blood transfusions. On follow-up they have been diagnosed with chronic hemolysis as evidenced by reticulocytosis and indirect hyperbilirubinemia. Molecular analysis revealed combined missense mutations in exons 12 and 13. The first mutation was 1376 G→T predicting 459 Arg→Leu (known as G6PD Canton) and the second one was 1502 T→G predicting 501 Phe→Cys. We designated the resulting novel G6PD variant, G6PD Bangkok Noi.     

Molecular basis of G6PD deficiency in India

G6PD deficiency has been reported from India more than 30 years ago and about 13 variants have been characterized biochemically. Here, we report the results of an epidemiological study investigating G6PD deficiency and the mutations among 14 heterogenous populations of India. Of the 3166 males tested, 332 (10.5%) were found to be G6PD-deficient and the prevalence rate varied from 5.7% to 27.9% in the different population groups. Molecular characterization revealed that G6PD Mediterranean (563 C-->T) was the commonest (60.4%) deficient variant followed by G6PD Kerala-Kalyan (949 G-->A; 24.5%) and G6PD Orissa (131 C-->G; 13.3%). G6PD Mediterranean had a more widespread distribution as compared to G6PD Kerala-Kalyan and G6PD Orissa and was associated with both 1311 C and 1311 T polymorhism. G6PD Mediterranean was found to have significantly lower red cell enzyme activity and more severe clinical manifestations than the other two. G6PD Chatham (1003 G-->A) with undetected red cell enzyme activity and G6PD Insuli (989 G-->A) with normal G6PD activity were very rare in the Indian population. The absence of a large number of mutations causing G6PD deficiency points to the fact that the genetic diversity of these populations is considerably lowered than expected.     

A new mutation responsible for severe G6PD deficiency in two ethnic Chinese with different clinical presentations: determination by a direct PCR sequencing technique.

To test the hypothesis that clinical manifestation in G6PD deficiency correlates with a molecular lesion, we investigated the G6PD gene of two Chinese Americans both of whom had G6PD deficiency, but who manifested different clinical presentations. In this study, we have developed a direct PCR sequencing protocol to examine the human G6PD gene. By using optimized PCR conditions with internal primers, we were able to amplify a 4.2 kb DNA fragment (covering exon 3 through 13 of the G6PD gene) of consistently high quality. From this we were then able to generate high quality single-stranded DNA templates by asymmetric PCR for subsequent sequencing. We also overcame the crossband problem by using internal primers, high temperature reaction with Taq I DNA polymerase, and/or sequencing with gene 32 protein. We could consistently amplify exons 1 and 2 despite their high G/C content by substituting 75% of dGTP with deoxy-7-deaza-guanosine triphosphate. By using this novel approach, we have identified a new mutation at cDNA position 1376 from G to T, which causes substitution of Leu for Arg at amino acid position 459. This mutation has not been reported in other ethnic groups. It is the only genetic defect in the coding regions of the G6PD gene of these two G6PD deficient individuals. We speculate that in addition to a defect in the G6PD gene, other factors also play a role in the clinical manifestation of G6PD deficiency.