Editor—Galactosaemia is an autosomal recessively inherited metabolic disorder caused by a defect in the galactose-1-phosphate uridyltransferase (GALT) enzyme. Absence or severe reduction of GALT activity results in classical galactosaemia (G/G) while an approximately half reduction of enzyme activity leads to the Duarte variant of galactosaemia (D/D). Mutation Q188R was found to be the most common molecular defect among classical galactosaemia patients,1 whereas N314D was predominantly detected in Duarte galactosaemia patients.2 In recent studies, the Duarte (D2) allele with 50% of normal GALT activity and the Los Angeles (D1) allele with 110-130% of normal GALT activity were characterised as having nucleotide alterations in addition to N314D.3 A study of the GALT gene from 31 unrelated galactosaemia families and from 504 control subjects is reported.

Thirty three patients and their relatives from the Czech and Slovak Republics were investigated (100% of all known galactosaemia patients). All families were white. There is no galactosaemia newborn screening in our country. Clinical onset of classical galactosaemia began in all patients in the neonatal period. The patients were identified with typical symptoms of classical galactosaemia (vomiting, failure to thrive, icterus, sepsis, hepatosplenomegaly, cataracts) and diagnosed by erythrocyte GALT assay (residual GALT activity less than 3% of the control value).4 A total of 504 non-galactosaemic subjects from the Czech Republic, including healthy donors and parents of α-1-antitrypsin deficiency patients, were used as a control group for population screening of the Duarte (D2) and Los Angeles (D1) alleles.

Mutation analysis was performed for the 5′ upstream region and the whole coding region with flanking intronic sequences of the GALT gene using PCR/digestion, denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HA), and sequencing methods. A total of 11 sequence variations in six mutated alleles was found. The two most common molecular defects were the mutations Q188R (45.2%) and K285N (27.5%). Two novel mutations in the coding region of the GALT gene, Y209S (3.2%) and 2142delGCC (1.6%), were detected; both were associated with severe reduction of enzyme activity. The previously described mutation L195P was found in exon 7 on three mutant alleles (4.8%). An unusual molecular genotype was observed in three classical galactosaemia alleles (4.8%), with six variations from the normal nucleotide sequence presented in cis (V151A, N314D, −119del4, 1105G→C, 1323G→A, and 1391G→A). A novel deletion of four GTCA nucleotides in the 5′ promoter region, in a position 119 nucleotides upstream from the initiation codon (−119del4), was found in Duarte (D2) alleles (fig 1, above), in addition to N314D, 1105G→C, 1323G→A, and 1391G→A. The deletion abolished aDdeI restriction site in the amplification created restriction site (ACRS) detection system (fig 1, below). In addition, during analysis of the 5′ promoter region, a discrepancy was found between published5 and detected sequences (fig 2).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

(Above) Sequence analysis of the 5′ upstream region of the GALT gene. Partial sequence ladders from the –119del4 homozygote (left) and from a control (right). Arrows indicate mutation site within the sequence ladder. (Below) Restriction enzyme analysis of the –119del4 mutation. The DNA samples were amplified using the primers PROM del4: 5′-CAGGGCAGCCCAGTCACTCA-3′ and PROM B: 5′-GCGTTGCTGAGGATCGGTTC-3′ followed by digestion by DdeI. Lane 1: homozygous wild type. Lanes 2, 4, 5: heterozygous. Lane 3: homozygous mutant.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

(A-C) Diagram of part of the GALT promoter sequence. Underlined nucleotides were incorrect in originally published sequence.5 The arrow over the nucleotides in (B) denotes the position of the PROM del4 primer. The shaded box denotes the position of the nucleotides deleted in the Duarte (D2) allele. The number over the grey box denotes the nucleotide position upstream from the initiation codon. (A) Originally published sequence, Leslie et al.5 (B) Corrected sequence of normal allele. (C) Duarte (D2) allele sequence.

Using PCR/restriction digestion assay, we screened a sample of 1008 control alleles, obtained from 504 subjects, for N314D plus accompanying intron and exon variations. The control subjects had no clinical symptoms of galactosaemia (biochemically and electrophoretically untested). N314D was found on 82 of 1008 (8.2%) different control alleles examined. From these, 54 (5.4%) were Duarte (D2) alleles contained in cis N314D plus –119del4, 1105G→C, 1323G→A, and 1391G→A. Twenty eight (2.8%) were Los Angeles (D1) alleles carrying in cis mutation N314D plus the silent mutation L218L. From previously obtained results,2 6 as well as the study presented here, it is evident that the Duarte (D2) and Los Angeles (D1) alleles are widespread among various populations. In both Czech galactosaemic V151A + Duarte (D2) and control Duarte (D2) alleles, the promoter deletion –119del4 and the intronic variations 1105G→C, 1323G→A, and 1391G→A were in linkage disequilibrium with N314D. Based on these results, we assume that the Duarte (D2) allele contains the promoter deletion –119del4 and the three intron substitutions, 1105G→C, 1323G→A, and 1391G→A, together with N314D. N314D appears to be an ancient genetic variant of the GALT gene, a background on which several other sequence variations were created.

The mechanism of partial GALT activity impairment in the Duarte alleles still remains to be fully understood. Two possible explanations have been described. Lai et al 7showed that the N314D mutation reduces the biological stability of the GALT dimeric protein in human lymphoblastoid cell lines; they stated, however, that no nucleotide changes other than N314D had been found in the GALT genes they studied. This finding is, though, in conflict with results obtained from the yeast expression system, in which the N314D subunit dimerises well both with wild type GALT and with itself. The reduced stability was not seen when N314D containing GALT protein was overexpressed via exogenous promoters in yeast.8 On the other hand, Podskarbi et al 9reported that, besides N314D, Duarte alleles (D2) carry two “intronic mutations”, 1105G→C and 1391G→A. They suggested that these intron alterations might be “regulatory mutations” involved in regulation of GALT gene expression.

In the present work, we have described a new DNA alteration on Duarte alleles, the deletion of four nucleotides (GTCA) in the 5′ promoter region of the GALT gene (−119del4). There is a high probability that this deletion is located in the transcription factor binding region. For this reason a computer search for potential regulatory DNA elements in the area of the deletion was performed.10 TwoHomo sapiens binding factors (activator proteins AP1 Q2 and AP1 Q4), which lose their binding motifs in Duarte (D2) alleles, were found. We conclude that the –119del4 promoter mutation is perhaps the main factor in Duarte allele enzyme activity reduction caused by a decrease in the synthesis of mRNA. This hypothesis will be tested further; however, Shin et al 6 reported that in competitive RT-PCR, the RNA level from homozygous Duarte (D2) cultured human lymphocytes was lower than that obtained from control cultured human lymphocytes.