Human G6PD variant structural studies: Elucidating the molecular basis of human G6PD deficiency

Glucose-6-phosphate dehydrogenase deficiency is by far the most prevalent human enzymopathy and is gener�ated by a series of point mutations in the X-linked gene encoding G6PD. The severity of the deficiency relies on the various mutational sites in the gene, affecting the protein structure and function in at least two ways: by disrupting the entire protein fold or by altering the functional groups. Thus, the modified enzyme should be identified structurally and functionally to recognize the sequelae of each mutation. Understanding the molecular basis of G6PD deficiency is also essential to determine how mutations influence enzyme structure, stability, and activity. In characterizing 34 G6PD variants selected from Class I, II, and III, we reviewed and compared structural and molecular characterizations. These studies have shown that these mutations can influence the G6PD enzyme's local and global stability by changing the features of the mutant amino acids or by modifying their interactions (lost, increased, or decreased distances). Furthermore, the relationship between the changes in the enzyme structure and the severity of the disease was also reviewed. Overall, their results showed that Class I had the strongest influence on the protein's stability, activity, and function, which correlated with chronic non�spherocytic hemolytic anemia. Furthermore, there have been no drugs available to treat G6PD deficiency until now

LEP G2548A polymorphism is associated with increased serum leptin and insulin resistance among T2DM Malaysian patients

Background: Type 2 diabetes mellitus (T2DM) is a chronic metabolic syndrome that is rapidly increasing across the world, especially in Malaysia. Leptin plays a vital role in the regulation of metabolism through its effect on peripheral tissues. G2548A polymorphism in the LEP gene promoter has been associated with insulin resistance, leptin, and type 2 diabetes mellitus across different population, but has not been inclusively reported within the Malaysian population. Objective: Thus, our study aimed to investigate the impact of G2548A polymorphism on serum leptin levels and insulin resistance among Malaysian T2DM patients. Methods: This case-control study involved 150 T2DM patients and 150 non-diabetic volunteers from ethnic Malays, Chinese and Indians. Genotyping of G2548A polymorphism was carried out using PCR-RFLP. Serum leptin and insulin levels were determined via ELISA. ANOVA and Chi-square tests were used to determine the distribution of genotypes and allelic frequencies based on serum leptin and insulin levels. Results: Frequency of AA genotype and A allele of G2548A variant were significantly (P < 0.05) higher in T2DM patients of Malay and Indian ethnicities (4%, 35%, and 36%, 57%, respectively) as compared to the control groups (0%, 22%, and 18%, 35%, respectively). Fasting serum leptin levels were significantly (P < 0.001) higher in T2DM patients compared to non-diabetic subjects (166.78 pg/ml, 101.94 pg/ml, respectively). Additionally, elevated serum leptin, insulin levels, and BMI in diabetic patients were found to be associated with the AA genotype of this variant, compared to GG, and GA genotypes (P < 0.05). Conclusion: Our findings suggest a significant association between G2548A polymorphism among Malaysian T2DM subjects, particularly among Malay and Indian ethnic groups. Moreover, the A allele frequency of the G2548A variant significantly increased the risk of T2DM and is significantly associated with increased serum leptin, insulin levels, and elevated BMI

Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Background: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. Methods: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. Results: Based on phenotypic testing, the prevalence of G6PD deficiency (T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. Conclusions: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population

Application of microsatellite dna markers for genetic diversity analysis in wild and domesticated stock of macrobrachium rosenbergii / Nurriza Ab. Latif

Ten microsatellite loci (Mbr-1, Mbr-3, Mbr-4, Mbr-5, Mbr-7, Mbr-8, Mbr-10a, Mbr-10b, UVC-807 and UVC-817) were used to assess the genetic diversity and makeup of different populations of Macrobrachium rosenbergii in Malaysia. A total of 120 individuals from four hatcheries and 116 individuals from four wild populations were studied. The samples were Sarawak River (SRWK), Terengganu River (TRGN), Timun River (NSBL), Kedah River (KDH), Thailand (THAI), National Prawn Fry Production and Research Centre (NAPFRE), Mun‟s Aquaculture (HatA) and Wong‟s Aquaculture (HatB). Allele sizes of all samples were scored using the ABI 3100 Genetic Analyzer. A total of 161 alleles were detected at 10 loci across the eight populations. All domesticated and wild populations demonstrated relatively high genetic variation with an average of 9.00 to 13.10 alleles per locus (A) and observed heterozygosities (Ho) ranging from 0.5833 to 0.8000. All the studied populations deviated from Hardy–Weinberg equilibrium proportions at a number of loci. Genetic distance computed by Nei (1978) denotes that the shortest genetic distance was between KDH and NSBL (D=0.2087) while the greatest was between HatA and NAPFRE (D=0.9333). Analysis of molecular variance (AMOVA) revealed significant differentiations in all populations with moderate FST values of between 0.0311 and 0.1438. Phylogenetic analysis depicted a close genetic relationship of M. rosenbergii between HatB and NSBL while samples from HatA diverged and was not clustered with any populations though the three populations originated from the same area. Microsatellites analysis revealed high levels of genetic variation and genetic differentiation among all the populations tested. Regular monitoring on genetic diversity and structure of M. rosenbergii is needed for genetic improvement and management programmes

A Bibliometric Analysis and Review of Pullulan-Degrading Enzymes—Past and Current Trends

Starch and pullulan degrading enzymes are essential industrial biocatalysts. Pullulan-degrading enzymes are grouped into pullulanases (types I and type II) and pullulan hydrolase (types I, II and III). Generally, these enzymes hydrolyse the α-1,6 glucosidic bonds (and α-1,4 for certain enzyme groups) of substrates and form reducing sugars such as glucose, maltose, maltotriose, panose or isopanose. This review covers two main aspects: (i) bibliometric analysis of publications and patents related to pullulan-degrading enzymes and (ii) biological aspects of free and immobilised pullulan-degrading enzymes and protein engineering. The collective data suggest that most publications involved researchers within the same institution or country in the past and current practice. Multi-national interaction shall be improved, especially in tapping the enzymes from unculturable prokaryotes. While the understanding of pullulanases may reach a certain extend of saturation, the discovery of pullulan hydrolases is still limited. In this report, we suggest readers consider using the next-generation sequencing technique to fill the gaps of finding more new sequences encoding pullulan-degrading enzymes to expand the knowledge body of this topic

A Bibliometric Analysis and Review of Pullulan-Degrading Enzymes&mdash;Past and Current Trends

Starch and pullulan degrading enzymes are essential industrial biocatalysts. Pullulan-degrading enzymes are grouped into pullulanases (types I and type II) and pullulan hydrolase (types I, II and III). Generally, these enzymes hydrolyse the &alpha;-1,6 glucosidic bonds (and &alpha;-1,4 for certain enzyme groups) of substrates and form reducing sugars such as glucose, maltose, maltotriose, panose or isopanose. This review covers two main aspects: (i) bibliometric analysis of publications and patents related to pullulan-degrading enzymes and (ii) biological aspects of free and immobilised pullulan-degrading enzymes and protein engineering. The collective data suggest that most publications involved researchers within the same institution or country in the past and current practice. Multi-national interaction shall be improved, especially in tapping the enzymes from unculturable prokaryotes. While the understanding of pullulanases may reach a certain extend of saturation, the discovery of pullulan hydrolases is still limited. In this report, we suggest readers consider using the next-generation sequencing technique to fill the gaps of finding more new sequences encoding pullulan-degrading enzymes to expand the knowledge body of this topic

The association of LEPR Q223R polymorphism with type 2 diabetes mellitus in Malaysia

Background: Type 2 diabetes mellitus (T2DM) is a major global public health problem that leads to increased risk of morbidity and mortality due to its complications. Leptin receptor plays a crucial role in regulating glucose metabolism and insulin sensitivity. Thus, mutation in the leptin receptor gene might play a role in the pathogenesis of T2DM. Objective: Our study aimed to evaluate the association of the Q223R polymorphism in the LEPR gene with Malaysian T2DM patients. Methods: A case-control study was focused on the three major ethnic groups of Malaysian population (100 Malays, 100 Chinese, and 100 Indians). Genotyping analysis of LEPR Q223R polymorphism was carried out by PCR-RFLP in 150 T2DM patients and 150 non-diabetic subjects as control. Serum insulin and leptin levels were determined using ELISA. A homeostasis model assessment-insulin resistance (HOMA-IR) and BMI were also calculated. Results: T2DM patients had significantly (P > 0.001) higher serum leptin levels as compared to the non- diabetic volunteers (166.78 pg/ml, 101.94 pg/ml, respectively). The frequency of AG genotype of LEPR Q223R variant was significantly higher in T2DM patients as compared to the control group (58.66% vs. 42%, χ2 = 8.75, p = 0.013). The A allele frequency was significantly higher in T2DM patients than the non-diabetic individuals (36.66% and 29%, respectively, P = 0.046). Furthermore, there were markedly elevated serum leptin, insulin, HOMA-IR, and BMI in diabetic patients with GG genotype of this variant as compared to AA, and AG genotypes (P < 0.05). Conclusion: Our findings demonstrated a significant association between LEPR Q223R polymorphism and T2DM in Malaysian subjects, particularly in Malay and Chinese ethnics, but not for Indian ethnic. This study suggested the A allele frequency of Q223R variant significantly increases the risk of T2DM in Malaysian population. Likewise, the polymorphism of Q223R in the LEPR gene is also associated with markedly increased serum insulin, leptin, HOMA-IR, and elevated BMI in T2DM patients

Additional file 1 of Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Additional file 1: Figure S1. Primers used in (A) multiplex HRM for the detection of 15 G6PD mutations and (B) G6PD gene sequencing. Table S1. Primers used in multiplex HRM assays. Table S2. Primers used for site-directed mutagenesis. Figure S2. The frequency distribution of G6PD enzyme activity in (A) males and (B) females. Figure S3. Box plot of G6PD activity for each variant among (A) malaria-positive males, (B) malaria-positive females, (C) malaria-negative males and (D) malaria-negative females. Figure S4. Secondary structure analysis of G6PD variants by circular dichroism. Table S3. Melting temperature (Tm) values of recombinant G6PD proteins by thermal shift assay. Mutations were ranked in order of stability, from most stable to least stable. Table S4. Thermal inactivation of G6PD variants as reported by T1/2. Mutations were ranked in order of stability, from most stable to least stable. Table S5. Stability of G6PD variants in the presence of Gdn-HCl as reported by C1/2. Mutations were ranked in order of stability, from most stable to least stable. Table S6. Susceptibility of G6PD variants to trypsin digestion. Mutations were ranked in order of stability, from most stable to least stable. Table S7. Structural characteristics of the dimer and tetramer interfaces (t = 100 ns). Table S8. Average values of the trajectory analyses performed on the WT and variants. Figure S5. Ligand binding pocket occupancy heatmap indicating the presence (orange) and absence (turquoise) of hydrogen bonds (t = 100 ns). Figure S6. Superimposition and structural deviations of the simulated variants against the WT (red) at the mutation site, dimer and tetramer interfaces (t = 100 ns). (A) Gaohe, (B) Valladolid, (C) Canton, (D) Viangchan, (E) Gond, (F) Gaohe + Viangchan, (G) Valladolid + Viangchan, and (H) Canton + Viangchan