Detection of novel mutations in galactose-1-phosphate uridyltransferase (GALT) gene in the galactosemia patients by temporal temperature gradient gel electrophoresis (TTGE)

Classical galactosemia caused by deficiency of galactose-1-phosphate uridyl - transferase (GALT) is characterized by acute symptoms of hepatocellular dysfunction, sepsis, cataracts and failure to thrive. A restriction of dietary galactose reverses these complications immediately, however, the long-term complications, such as mental retardation and ovarian failure remain as significant problems in most galactosemia patients. The human GALT gene has been mapped to chromosome 9 pl3 and is organized into 11 exons and 10 introns spanning four kilobases (kb) DNA. The predicted protein encoded by the GALT gene is 379 amino acids in length. More than thirty mutations distributed across all the exons have been identified thus far in affected individuals. A single missense mutation, in codon 188 (Q 188R), accounts for over 60% of galactosemia alleles in Caucasian patients, while the S 135L mutation is the most common in African- American patients. This work was undertaken in order to improve our understanding of the molecular basis of galactosemia. Conditions for the temporal temperature gradient gel electrophoresis (TIGE) method of mutation screening were established for exons 2 and 10 of the GALT gene following amplification of the regions of genomic DNA of galactosemia patients by polymerase chain reaction (PCR). Nucleotide sequence analysis was performed on- fragments that migrated aberrantly in the TIGE assay, and the sequences were compared to the previously published GALT sequence. Of the galactosemia patients screened by TIGE of exons 2 and 10, three were shown to have nucleotide alterations. These changes were confirmed by nucleotide sequence analysis. The alterations were: (1) substitution of an arginine codon for a leucine codon at position 51 (R51L) in exon 2 that was confirmed by the absence of a Hae III site; (2) a silent (third position) change in codon 338; and (3) the deletion of a C at nucleotide position 1047 in codon 349 (~1047C) in exon 10, which causes a frameshift and results in a predicted early termination of translation at amino acid position 358. In the course of this study, the sensitivity of TIGE and of heteroduplex analysis were compared and TIGE was found to be more sensitive as a screening technique for mutations in the GALT gene. All of the three alterations identified in this work are novel, and the two of probable biological significance occurred in highly conserved domains of the GALT protein.