Abstract
Iraq contains many diseases that have never been counted or examined, including diseases related to food, which has deteriorated in recent years, and has rapid and direct impact especially on the children category, one of these diseases is galactosemia. Classical galactosemia, deficiency of galac tose-1-phosphate uridyltransferase GALT, is characterized by acute symptoms of hepatomegaly, jaundice, sepsis, cataract, vomiting, and diarrhea and growth retardation. Our previous molecular study showed that the most common mutation of the GALT gene is a missense mutation of Q188R (replacement of glutamine-188 by arginine in exon 6 and N314D mutation replacement of aspargen-314 by aspartic acid) in exon 10. The aim of this study was to determine the possibility of diagnosing galacatosemia, and to search for galactosemia mutation Q188R and N314D in Iraqi population. Blood samples were collected from babies admitted to the children's hospitals in Mosul City depending on the clinical symptoms of disease and then serum was taken. Measuring the Galactose-1-Phosphate uridylytransferase GALT enzyme activity and galactose -1- phosphate in serum by ELISA technique was done. DNA samples were analyzed by the polymerase chain reaction followed by digestion with restriction endonuclease HpaΙΙ and AvaΙΙ for Q188R and N314D mutation. The results showed a significant decrease in the level of the GALT enzyme in children with galactosemia 21.7 ± 0.45 and among non-diagnosed children 79.93 ± 1.44 compared with control group 160.33 ± 0.93 as well as a significant decrease in the level of the enzyme among mothers 20.5 ± 1.92 was observed. Gal-1-P level was significantly higher in the cases than that of the control group, while that of the not diagnosed children and mothers groups showed inconsistent difference. Also the result showed absence allele frequency for Q188R mutation and present allele frequency for N314D mutation in Iraqi population. In conclusions It is possible to depend on measurement of Galactose-1-Phosphate as indicator in the diagnosis of Galactosemia in newborn, the main mutation in GALT gene causes galactosemia is N314D in Iraqi population.
Highlights
Galactosemia is an autosomal recessive disorder caused by a deficiency of one of the enzymes involved in the utilization of dietary galactose; galactokinase EC 2.7.1.6, galactose-1-phosphate uridyl transferase EC 2.7.7.12, or uridinediphosphate galactose-4-epimerase EC 5.1.3.2 (18)
The results showed a significant decrease in the level of the GALT enzyme in children with galactosemia 21.7 ± 0.45 and among non-diagnosed children 79.93 ± 1.44 compared with control group 160.33 ± 0.93 as well as a significant decrease in the level of the enzyme among mothers 20.5 ± 1.92 was observed
In the present study a significant decrease in the level of the enzyme GALT in children with galactosemia compared with healthy as well as a significant decrease in the level of the enzyme in the mothers and non-diagnosed children compared with the control group these results agreed with other studies [7,12], which demonstrated a decrease of GALT enzyme level in patients with galactosemia compared with healthy group
Summary
Galactosemia is an autosomal recessive disorder caused by a deficiency of one of the enzymes involved in the utilization of dietary galactose; galactokinase EC 2.7.1.6, galactose-1-phosphate uridyl transferase EC 2.7.7.12, or uridinediphosphate galactose-4-epimerase EC 5.1.3.2 (18). The biochemical basis of both the acute and chronic symptoms is not well understood, and there is no correlation between outcome and genotype, residual GALT activity or the red blood cell level of galactose-1-phosphate, which is used to monitor treatment [1,4]. The Q188R allele is associated with essentially no activity in human erythrocytes or lymphoblasts or in yeast engineered to express the human alleles [3,6] Another common mutant allele of the GALT genome is an A-to-G transition in bp 2744 of exon 10, which results in the substitution of an acidic aspartate (D), for an asparagines (N) at codon 314 (N314D) [7]. PCR was carried out in a final volume of 20 μl, with pri mex from promega, The following cycling conditions were used: cycle 1: 95for 5 min, 65° for 1 min, and 72 for 1 min; cycles 2-34: 95 for 45 s, 65° for 1 min, and 720 for 1 min; cycle 35: 95 for 45 min 65 for 1 min, and 72 for 8 min [6,11] The PCR products were digested with restriction endonucleases HpaII and SinI, as recommended by the manufacturer (Promega) for 3 hours and were analyzed by electrophoresis through a 2% agarose gel [6,11]
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