Birgit S. Gathof

Molecular characterization of Duarte-1 and Duarte-2 variants of galactose-1-phosphate uridyltransferase

SummaryThe N314D polymorphism was found in two different alleles of the galactose-1-phosphate uridyltransferase (GALT) gene, Duarte-1 (D1) and Duarte-2 (D2). Although both variants have identical electrophoretic mobility and isoelectrofocusing points, the galactose-1-phosphate uridyltransferase (GALT) activity varies: D1 alleles showed 110–130% of the normal RBC activity, but D2 alleles only 40–50%. We found that D1 alleles also carried a silent mutation in exon 7 (L218L) in addition to N314D. In contrast, besides N314D, D2 alleles carried two regulatory mutations, G1105C and G1391A, in introns D and E, respectively. In normal and Q188R alleles none of the above four mutations coexisted. However, some galactosaemia alleles with mutations other than Q188R, such as W316X and E340X of exon 10, also carried the N314D mutation. The W316X alleles existed in cis with the intron mutations (G1105C and G1391A), whereas those with E340X are in cis with L218L. In all cases examined, the intron mutations were not found in D1 alleles and no D2 alleles had the silent mutation of L218L. These results suggest that the decrease in the GALT activity in D2 may be due to regulation of the GALT gene expression. The G1105C site may be critical to the function of erythroid transcription factor NF-E1, since it flanks the core consensus sequence for one of its binding sites. The G1391A mutation may affect another cis-acting regulatory sequence. Alternatively, both mutations may be involved in an aberrant splice processing, which possibly results in a low level of correctly spliced mRNA.

Characterization of two stop codon mutations in the galactose-1-phosphate uridyltransferase gene of three male galactosemic patients with severe clinical manifestation

Classical galactosemia, which is caused by deficiency of galactose-1-phosphate uridyltransferase, is characterized by acute problems of hepatocellular dysfunction, sepsis, cataracts and failure to thrive. Galactose limitation reverses these symptoms immediately; however, the long-term complications, such as mental retardation and ovarian failures are major problems in most of these patients. In order to investigate the molecular basis for phenotype variation in galactosemia, we have screened the most common mutation in the GALT gene, Q188R. We have further examined those patients who are heterozygous for Q188R or negative for this mutation by SSCP analysis and direct sequencing. In three male patients, we have identified, for the first time, two stop-codon mutations in the GALT gene, G212X (exon 7) and E340X (exon 10). Two patients of 8 and 28 years of age, respectively, who are compound heterozygotes for Q188R and G212X, have severe mental retardation and their general clinical condition is more severe than that of patients with missense mutations. The third patient, who is 8 years of age and who is homozygous for E340X, the N314D polymorphism and a silent substitution L218L, presents with a relatively normal physical and mental condition to date.

Uric acid levels in southern Germany in 1989. A comparison with studies from 1962, 1971, and 1984.

SummarySince 1962 our group has performed four studies on uric acid values in blood donors in southern Germany (Bavaria). Uric acid levels in men have increased over the years, from 4.86 mg/dl in 1962 to 6.00 mg/dl in 1971, 5.60 mg/dl in 1984, and 5.90 mg/dl in 1989. Levels in women have increased slightly, from 4.05 mg/dl in 1962 to 4.35 mg/dl in 1971, 4.10 mg/dl in 1984, and 4.16 mg/dl in 1989. Women aged 51 to 60 years had significantly higher uric acid levels than those in the fourth decade. In women treated with oral contraceptives uric acid levels were significantly lower than in other women of the same age.Hypouricemia (uric acid levels ≤2.0 mg/dl) was observed in three women, none of whom had a history of medication.Hyperuricemia exists when uric acid levels are ≥6.5 mg/dl. In 1989 2.6% of the female blood donors and 28.6% of the males were hyperuricemic, with an increased risk of gout, nephrolithiasis, and nephropathy.

Mutations in the galactose-1-phosphate uridyltransferase gene of two families with mild galactosaemia variants

SummaryClassical galactosaemia, deficiency of galactose-1-phosphate uridyltransferase (GALT), is characterized by acute symptoms of hepatomegaly, jaundice, sepsis, cataracts and growth retardation. Treatment with dietary galactose restriction corrects these complications immediately; however, most of these children develop long-term complications of verbal dyspraxia, mental retardation and ovarian failure. 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 approximately 60–65% of the German galactosaemic population. The coding region of GALT was amplified by the polymerase chain reaction from genomic DNA of classical galactosaemic individuals, who are negative or heterozygous for Q188R, and was further characterized by direct sequencing. Three new disease-causing mutations, two missense and a stop codon mutation, were identified in three patients from two families with mild galactosaemic variants: firstly R67C, replacement of arginine-67 by cysteine and W316X, the stop codon at tryptophan-316 in one male; secondly A330V, replacement of alanine-330 by valine in two female siblings. In the first family the patient was also heterozygous for the polymorphism N314D and in the second family both girls were compound heterozygotes for Q188R and A330V. All three galactosaemic individuals have a considerable amount of the residual GALT activity in RBC and the galactose-1-phosphate (GALP) level decreased much faster on treatment than that of other galactosaemic patients with missense mutations such as Q188R. The clinical and biochemical data of these patients were much more favourable in comparison with those of two female galactosaemic individuals, one homozygous for L195P and the other compound heterozygous for Q188R and L195P. These three missense mutations (R67C, L195P and A330V) also occur in highly conserved regions. These observations suggest that the phenotypic variation in galactosaemic individuals may be due to different molecular aetiologies.

Three missense mutations in the galactose-1-phosphate uridyltransferase gene of three families with mild galactosaemia

Classical galactosaemia caused by deficiency of galactose-1-phosphate uridyltransferase (GALT) is characterized by acute symptoms of hepatocellular dysfunction, sepsis, cataracts and failure to thrive. Galactose limitation reverses these complications immediately, however, most of these children have a long-term complication of verbal dyspraxia, mental retardation and ovarian failure. The GALT gene was cloned and several mutations including the common Q188R have been reported. In this study the coding region of GALT was amplified by polymerase chain reaction from genomic DNA of classical galactosaemic individuals and characterized by direct sequencing of the products. Three missense mutations were identified in three patients with a mild galactosaemic variant: (1) replacement of threonine-138 by methionine (T138M); (2) replacement of arginine by tryptophan (R259W); and (3) replacement of threonine by alanine (T350A). All three galactosaemic individuals, one girl and two boys, have varying degrees of residual GALT activity in RBC and their galactose-1-phosphate levels decreased much faster than in other galactosaemic patients. These misense mutations occur in regions that are not highly conserved domains.ConclusionThe study of the molecular basis related to the phenotype variation may indeed help to prognosticate the outcome of patients with classical galactosaemia.

Benzbromarone and Fenofibrate are Lipid Lowering and Uricosuric: A Possible Key to Metabolic Syndrome?

Patients with hyperlipidemia often suffer from hyperuricemia and conversely. This is proven in many studies, e. g. our laboratory studied the levels of uric acid, cholesterol and triglycerides in German blood donors (Gresser et al., 1990; Gathof et al., 1991). 68 % of the men and 57 % of the women showed cholesterol levels of 200 mg/dl and more. Levels above 250 mg/dl were found in 25 % of the men and 15 % of the women. Hyperuricemia occured in 28.6 % of men and 2.6 % of women. There was a statistically significant correlation (p<0.001) between the level of uric acid and cholesterol respectively triglycerides. After elimination of the factor age the correlation coefficients dropped, especially in women, but still remained significant.

Identification of a splice mutation at the adenine phosphoribosyltransferase locus in a German family

SummaryWe examined the molecular basis of adenine phosphoribosyltransferase (APRT) deficiency in homozygous-deficient, identical twin brothers who were born to non-consanguineous German parents. DNA was isolated from blood, and the APRT gene was amplified by PCR, subcloned into M13, and sequenced completely. A single T insertion between bases 1831–1832 or 1832–1833 was identified. This alters the consensus sequence at the exon 4 — intron 4 splice donor site and leads to aberrant splicing. The same mutation has been described previously in two affected brothers from Belgium, and the Indianapolis group has also identified it in two other, unrelated Caucasian patients. Thus, this mutation may be a common cause of APRT deficiency in the Caucasian population.

A Splice Mutation at the Adenine Phosphoribosyltransferase Locus Detected in a German Family

Approximately 40 cases of type I adenine phosphoribosyltransferase (APRT) deficiency (complete deficiency) have been described. About 20 different mutations have been identified in patients with this disorder (Hidaka et al. 1987, Chen et al. 1991, Sahota et al. 1991). In Germany, five other cases of type I APRT deficiency have been found, two of Turkish and three of unknown ethnic origin. Our group has described a type I Caucasian family from southern Germany (Zollner and Gresser 1990). The aim of the present study was to investigate the molecular nature of the mutation in this family (Gathof et al. 1991a).

Importance of the Confounding Factors Age and Sex in the Correlation of Serum Uric Acid, Cholesterol and Triglyceride Levels

A correlation of serum uric acid (UA) to serum cholesterol (CHOL) and/or triglyceride (TRI) levels was discussed in several previous studies from other groups. A strong influence of age and sex on these parameters has also been reported. In this study we aimed to determine whether there is a real correlation between cholesterol, triglycerides and uric acid or a simulated one, created by the influence of sex and age.

The restriction enzyme Mse I applied for the detection of a possibly common mutation of the APRT locus

Eighteen different mutations of the APRT gene have been described in APRT-deficient patients (Sahota et al. 1992). From Europe and overseas different types of APRT mutations have been reported (Sahota et at. 1992). In a previous study we detected a T insertion in a family with APRT deficiency (Gathof et al. 1991). This T insertion alters the exon four-intron four splice donor site from T G : G T A A to T G : G T T A A or TG:GTTAA. Mse I recognizes the sequence TTAA and restricts it between the two thymidines.