Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by microarray-based assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy, affecting more than 200 million people worldwide. To date more than 123 mutations in the G6PD gene have been discovered, among which 12 point mutations are found in the Chinese. Setting up a simple and accurate method for detecting these mutations is not only useful for diagnosing G6PD deficiency under some circumstances that it is difficult to measure the activity of the enzyme, but also for studying the frequency of the G6PD genotypes. The purpose of this study was to develop a simple, inexpensive and accurate method for detecting these common mutations. Microarray-based assay was described in this study. Samples from 198 G6PD-deficient persons were investigated. The DNA sequencing data supported the results obtained by microarray-based assay. Thus, we concluded that the microarray-based assay is a rapid, simple, inexpensive, and accurate method for detecting the most common G6PD gene mutations among the Chinese. This method involves the selective amplification of human G6PD gene with specific oligonucleotide primers, fragmentation and labeling of PCR products, followed by hybridization with allele-specific oligonucleotide (ASO) probes on chip.     

Development and evaluation of a reverse dot blot assay for the simultaneous detection of six common Chinese G6PD mutations and one polymorphism

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. We developed and validated a reverse dot blot (RDB) assay for the rapid and simultaneous genotyping of six mutations (c.95A>G, c.871G>A, c.1004C>T, c.1024C>T, c.1376G>T and c.1388G>A), that were common mutations in the Chinese G6PD deficiency population, and one polymorphism (c.1311C>T). Reliable genotyping of wild-type and mutant genomic DNA samples was achieved by means of a test strip onto which allele-specific oligonucleotide probe lines are fixed in parallel. This method involves a multiplex PCR amplification of three fragments in the G6PD target sequence and a manual hybridization/detection protocol. The entire procedure starting from blood sampling to the identification of mutations requires less than 6 h. The diagnostic reliability of this reverse dot blot assay was evaluated on 207 pre-typed samples by using direct DNA sequence analysis in a blind study. The reverse dot blot typing was in complete concordance with the reference method. The reverse dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify common G6PD mutations in Chinese population.     

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.     

Rapid detection of six common Mediterranean and three non-Mediterranean alpha-thalassemia point mutations by reverse dot blot analysis

We describe the implementation of reverse dot blot (RDB) hybridization as a rapid nonradioactive method for the identification of six frequent globin gene point mutations in the Mediterranean population: alpha(Hph)alpha: alpha2 IVS I donor site GGTGAGG --> GG-----; alpha(NcoI)alpha: alpha2 initiation codon ATG --> ACG; alpha(TSaudi)alpha: alpha2Poly A signal AATAA --> AATAAG; alpha(Icaria)alpha: alpha2 termination codon TAA --> AAA (Ter --> LYS); alpha(CS)alpha: alpha2 termination codon TAA --> CAA (Ter --> gly); alphaalpha(NcoI): alpha1 initiation codon ATG --> GTG; and three alpha2 globin gene point mutations found in immigrants in Italy: alpha(T-Quongsze)alpha: alpha2 codon 12 CTG --> CCG (Leu --> Pro); alpha(Seal Rock)alpha: alpha2 termination codon TAA --> GAA (TER --> GLU); and alpha(Koyadora)alpha: alpha2 termination codon TAA --> TCA (TER --> SER). The method uses the principle of allele-specific oligonucleotide (ASO) hybridization, but it is a nonradioactive method and permits rapid and simultaneous typing of point mutations and small deletions.     

Glucose-6-phosphate dehydrogenase (G6PD) mutations and UDP-glucuronosyltransferase promoter polymorphism among G6PD deficient Kuwaitis

Screening of 1,080 Kuwaiti male blood donors for glucose-6-phosphate dehydrogenase (G6PD) deficiency revealed this condition in 70 (6.5%) individuals. Mutation analysis of all 70 G6PD deficient samples performed by PCR/RFLP and direct sequencing identified the 563C-->T (Mediterranean) in 72.9%, 202G-->A (A(-)) in 14.3%, 1003G-->A (Chatham) in 7.1%, and 143T-->C (Aures) in 1.4%. In 3 cases (4.3%) mutations remain unknown. Genotyping of all G6PD deficient samples for UDP-glucuronosyltransferase 1 (UDPGT1) gene promoter polymorphism revealed (ta)6/(ta)6 in 38.6%, (ta)7/(ta)7 in 15.7%, (ta)6/(ta)7 in 44.3%, and (ta)7/(ta)8 allele in 1.4% of cases. Thus, 4% of males in the Kuwaiti population have G6PD deficiency coexisting with low activity of the UDPGT1 promoter.     

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.     

G6PD gene mutations in India producing drug-induced haemolytic anaemia

We report 17 cases of glucose-6-phosphate dehydrogenase (G6PD) deficiency with drug-induced haemolytic anaemia. In most cases the drug involved was an antimalarial. However, we also found two cases in which other drugs could have been responsible for the haemolysis. The degree of severity of haemolysis differed in the individuals and most required multiple transfusions.     

Two distinct Indian G6PD variants G6PD Jamnagar and G6PD Rohini caused by the same 949 G-->A mutation

Earlier we have reported two G6PD variants viz.; G6PD Jamnagar and G6PD Rohini. The enzymes from both the variants showed altered biochemical properties with mild enzyme deficiency and were classified as unique Class III variants. G6PD Jamnagar was found to be associated with drug-induced hemolytic anemia whereas G6PD Rohini was picked up during a population survey. Subsequent molecular studies on the DNA from both the cases showed the presence of the Kerala-Kalyan (949 G-->A) mutation. Hence, this study besides supporting the fact that biochemically distinct variants could have the same mutation at the molecular level also highlights the importance of molecular characterization of G6PD variants.     

Assessing the value of CAN-gene mutations using MALDI-TOF MS

Purpose

To identify cancer-linked genes, Sjöblom et al. and Wood et al. performed a genome-wide mutation screening in human breast and colorectal cancers. 140 CAN-genes were found in breast cancer, which in turn contained overall 334 mutations. These mutations could prove useful for diagnostic and therapeutic purposes.

Methods

We used a MALDI-TOF MS 40-plex assay for testing 40 loci within 21 high-ranking breast cancer CAN-genes. To confirm mutations, we performed single-plex assays and sequencing.

Results

In general, the mutation rate of the analyzed loci in our sample cohort was very low. No mutation from the 40 loci analyzed could be found in the 6 cell lines. In tissue samples, a single breast cancer tissue sample showed heterozygosity at locus c.5834G>A within the ZFYVE26 gene (Zinc finger FYVE domain-containing gene 26).

Conclusions

Sjöblom et al./Wood et al. already showed that the vast majority of CAN-genes are mutated at very low frequency. Due to the fact that we only found one mutation in our cohort, we therefore assume that at the selected loci, mutations might be low-frequency events and therefore, more rarely detectable. However, further evaluation of the CAN-gene mutations in larger cohorts should be the aim of further studies.     

G6PD (AC)n and (CTT)n microsatellites in Mexican Mestizos with common G6PD African variants

Genotyping for the G6PD (AC)n and (CTT)n microsatellites in a sample of 58 Mexican Mestizos with common G6PD African variants was carried out. The second mutation that defines to the variants G6PD A(-202A/376G), G6PD Santamaria(376G/542T) and G6PD A(-376G/968C) very probably occurred on G6PD A(376G) chromosomes with the compound haplotypes, intragenic silent polymorphisms and microsatellites, Pvu-II/Pst-I/Bcl-I/Nla-III/(AC)n/(CTT)n: +/+/-/+/166 bp/195 bp, -/+/-/+/166 bp/201 bp, and -/+/-/+/166 bp/204 bp respectively. The structure of the repeat sequences for the AC-166 bp allele in the 3 variants was (TA)5(AA)1(TA)9(CA)10 whereas the repeat sequences for the CTT-195 bp, CTT-201 bp and CTT-204 bp alleles were (CTT)11(ATT)6, (CTT)7(ATT)12 and (CTT)7(ATT)13 in the first, second and third variants respectively. Genotyping for the G6PD microsatellites can be a useful tool with several applications.     

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.     

Molecular genetics of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene

Summary .In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain, we have analysed the G6PD gene in 11 unrelated Spanish G6PD-deficient males and their relatives by using the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with a direct PCR-sequencing procedure and PCR-restriction enzyme (RE) analysis. We have identified eight different missense mutations, six of which have been reported in previously described G6PD variants. In nine patients who had presented with acute favism we found the following mutations: G6PD A_^376G–202A (four cases)| G6PD Union^1360T (two cases), G6PD Mediterranean^563T, (one case) and G6PD Aures^143c (one case). In the remaining patient a novel A to G transition was found at nucleotide position 209 which has not been reported in any other ethnic group. This mutation results in a (70) Tyr to Cys substitution and the resulting G6PD variant was biochemically characterized and designated as G6PD Murcia. This new mutation creates a Bsp 12861 recognition site which enabled us to rapidly detect it by PCRRE analysis. In two patients with chronic non-spherocytic haemolytic anaemia (CNSHA) we found the underlying genetic defects, as had been noted previously, to be located within a cluster of mutations in exon 10. One of them had the T to C transition at nucleotide 1153, causing a (3 8 5) Cys to Arg substitution, previously described in G6PD Tomah. The other, previously reported as having a variant called G6PD Clinic, has a G to A transition at nucleotide 1215 that produces a (405) Met to He substitution, thus confirming that G6PD Clinic is a new class I variant.     

G6PD Avenches and G6PD Moosburg: biochemical and erythrocyte membrane characterization

Summary Two new G6PD variants with severe enzyme deficiency in Switzerland (G6PD Avenches, G6PD I) and in Germany (G6PD Moosburg, G6PD II) are described. One patient had suffered from severe postpartal hyperbilirubinemia, the other one presented with chronic hemolysis and remittent hyperbilirubinemia. Both variants showed diminished electrophoretic mobility, both variants were heat labile. The Michaelis-Menten constants K_M for glucose-6-phosphate and for NADP⁺ were normal. 2-Desoxy-glucose-6-phosphate was utilized by G6PD I in a higher and by G6PD II at a lower rate than by the normal enzyme. Desamino-NADP⁺ and galactose-6-phosphate were utilized by both variants at a normal rate. The electrophoretic separation of membrane proteins of G6PD II showed both in the presence and in the absence of 6-mercaptoethanol no difference concerning the formation of membrane protein aggregates between patient and normal control.     

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.     

Two new glucose-6 phosphate dehydrogenase (G6PD) variants associated with hemolytic anemia: G6PD Amman-1 and G6PD Amman-2

Two glucose-6-phosphate dehydrogenase (G6PD) variants were investigated. G6PD Amman-1 was partially purified from the red cells of a patient suffering from recurrent jaundice and spontaneous episodic attacks of severe hemolysis in the absence of oxidant drugs, infection, or fava beans. The enzymatic characteristics of G6PD Amman-1 were markedly reduced activity, fast eletrophoretic mobility, slightly increased km for NADP, normal km for G-6-P, normal heat stability, normal utilization of substrate analogues 2-deoxy G-6-P and deamino-NADP, and a monophasic pH curve with a peak at 8.5 to 9.3. The second variant, G6PD Amman-2, was partially purified from the red cells of a patient suffering from recurrent jaundice with episodic mild hemolysis caused by infection or unknown factors. G6PD Amman-2 characteristics were severely reduced activity, slow electrophoretic mobility, normal km for NADP, decreased km for G-6-P, decreased heat stability, increased utilization of substrate analogues, and a monophasic pH curve with a narrow peak at pH 9.5. The red cell level of reduced glutathione was markedly decreased with twofold increase in the activity of glutathione reductase in the patient with G6PD Amman-2.