Inborn errors of metabolism screening and profiling of amino acids, acylcarnitines and glucose-6-phosphate dehydrogenase deficiency in Bangladesh population

Abstract

Similar to other lower and lower middle income countries, Bangladesh is facing the accelerated demographic shift from communicable diseases to non-communicable diseases (NCDs) including genetic disorders (GDs). Approximately 10,000 single-gene disorders have been identified which affects millions of people worldwide. One of the major categories of phenotypically and genotypically heterogeneous group of genetic disorders are inborn errors of metabolism (IEM). Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is commonly used for the diagnosis of more than 30 IEM. Accurate and reliable diagnosis of IEM by quantifying amino acids (AAs) and acylcarnitines (ACs) using LC-MS/MS systems depend on the establishment of age-specific cut-off values of the analytes for the population under investigation. Thus, the first objective of the study was to (1) determine the age-specific cut-off values of AAs and ACs in Bangladesh and (2) validate the LC-MS/MS method for diagnosis of the patients with IEM. A total of 570 healthy participants were enrolled in this study. They were divided into 3 age groups, namely, (1) newborns (1-7 days), (2) 8 days–7 years, and (3) 8–17 years, to establish the age-specific cut-offs for AAs and ACs. In addition, 273 suspected patients with IEM were enrolled to evaluate the reliability of the established cut-off values. Among these patients, nine cases came out as screening positive by LC-MS/MS and seven cases were confirmed by urinary GC-MS analysis including 3 cases with phenylketonuria, 1 with methylmalonic acidemia, 1 with isovaleric acidemia, 1 with citrullinemia type II, and 1 with carnitine uptake defect. Two borderline positive cases with medium-chain acyl-CoA dehydrogenase deficiency were negative by urinary GC-MS analysis. The second objective of the study was to detect mutations in glucose-6-phosphate dehydrogenase (G6PD) gene that are responsible for G6PD deficiency in Bangladeshi individuals because G6PD deficiency is also very common like IEM in the Indian subcontinent. Among 121 clinically suspected patients, 12 (11 males and one female) patients were found to be G6PD deficient, suggesting the frequency of G6PD deficiency among suspected patients is 9.9%. Sanger sequencing revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon 6: Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) in the coding sequence in one sample. From the study, it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Bangladesh

The third objective of the study was to establish a real-time PCR-based approach called high-resolution melt (HRM) analysis as a rapid and reliable method for the detection of G6PD variants in heterozygous females. Although hemizygous males and homozygous females are easily detected by the conventional G6PD enzyme assay method, the heterozygous state can be missed by the conventional methods as the mosaic population of both normal and deficient RBCs circulate in the blood. Sixty-three clinically suspected females were evaluated for G6PD status using both enzyme assay and HRM analysis. Four out of sixty-three participants came out as G6PD deficient by the enzyme assay method, whereas the HRM approach could identify nine participants with different G6PD variants, namely one homozygous and eight heterozygous. In conclusion, along with the establishment of a validated LC-MS/MS method for the quantitation of amino acids and acylcarnitines from the DBS cards, the study also demonstrates the presence of predominant IEM in Bangladesh. In addition, this study is the first report for the underlying genetic defects of the G6PD gene that causes G6PD deficiency in the Bangladeshi population and the establishment of a rapid and reliable HRM-based method for identification of G6PD variants among heterozygous females in Bangladesh.