Fumarylacetoacetase mutations in tyrosinaemia type I.

Sixty-two hereditary tyrosinaemia type I (HT1) patients of various ethnic origins were classified clinically into acute, chronic, or intermediate phenotypes and screened for the 14 published causal mutations in the fumarylacetoacetase (FAH) gene. Restriction analysis of PCR amplified genomic DNA identified 74% of the mutated alleles. IVS12 + 5G --> A, predominant in the French Canadian HT1 patients, was the most common mutation found in 32 alleles in patients from Europe, Pakistan, Turkey, and the United States. IVS6-1G --> T, encountered in 14 alleles, was common in Central and Western Europe. There was an apparent "Scandinavian" 1009G --> A combined splice and missense mutation (12 alleles), a "Pakistani" 192G --> T splice mutation (11 alleles), a "Turkish" D233V mutation (6 alleles), and a "Finnish" or northern European W262X mutation (7 alleles). The remaining mutations were rare. Some of the mutations seem to predispose for acute and other for more chronic forms of HT1, but in our material no clearcut genotype phenotype correlation could be established.

Tyrosinemia type 1--complex splicing defects and a missense mutation in the fumarylacetoacetase gene.

Two mutations are reported in six tyrosinemia type 1 patients from northern Europe. In four patients, a G to A transition at nucleotide position 1009 (G1009-->A) of the fumarylacetoacetase (FAH) coding sequence caused aberrant splicing by introducing an acceptor splice site within exon 12, thereby deleting the first 50 nucleotides of this exon. The following exon-intron boundary was frequently missed, and a cryptic donor splice site within intron 12 caused a partial intron 12 retention of 105 bp. This point mutation alternatively gave a glycine 337 to serine substitution in instances of correct splicing. The mutation is rapidly detected by PvuII digestion of polymerase chain reaction (PCR)-amplified genomic DNA. Another mutation, g+5-->a in the intron 12 donor splice site consensus sequence (IVS12 g+5-->a), was found in five of the patients. This caused alternative splicing with retention of the first 105 nucleotides of intron 12, exon 12 skipping, and a combined deletion of exons 12 and 13. Rapid detection of this mutation is achieved by restriction digestion of PCR-amplified genomic DNA; a mismatch primer combined with the point mutation creates a Tru9I restriction site. One patient who was homozygous for the G1009-->A mutation had a chronic form of tyrosinemia. Three patients were combined heterozygotes for G1009-->A and IVS12 g+5-->a. Their clinical phenotypes varied from acute to chronic, indicating the impact of background genes and/or external factors on the presentation of tyrosinemia type 1.

Maternal age and duration of labor.

The computerized records of a population of 7214 women who were delivered during the period 1987-1991 were analysed. We studied the possible relationship of the duration of the first and second stages of labor to maternal age. In para 0, para 1 and para 2+ mothers we found an independent positive correlation between the second stage duration and maternal age. By multiple stepwise regression analysis maternal age turns out to be one of the most influential maternal characteristics of the second stage of labor. No correlation was found between maternal age and the duration of the first stage.

The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency.

Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.

Does ovarian stimulation for in-vitro fertilization induce a hypercoagulable state?

Effects on blood coagulation and fibrinolytic activity during ovarian stimulation for in-vitro fertilization (IVF) were examined in 12 women. Blood samples were taken prior to hormonal stimulation (days 2-3 of the menstrual cycle, mean serum oestradiol concentration 0.16 nmol/l) and the day after ovulation induction with human chorionic gonadotrophin (HCG) (days 10-12, mean serum oestradiol concentration 5.35 nmol/l). We measured whole blood clotting time, whole blood clot lysis time, plasma fibrinogen, factor VII and antithrombin III. The whole blood clotting time was slightly, but not significantly shortened after ovarian stimulation. A significant rise in plasma fibrinogen (P less than 0.001) and reduction in antithrombin III (P less than 0.001) were observed, whereas no change in factor VII was found. The blood fibrinolytic activity was significantly reduced as evaluated by an increase in the clot lysis time (P less than 0.02). These results indicate that ovarian stimulation for IVF may create a state of hypercoagulability.

A new cell culture assay for quality control in IVF.

A new, simple and sensitive bioassay for quality testing in IVF is described. This bioassay (Medi-Cult Hybritest, GEA Ltd, Biotech Division, Hvidovre, Denmark) is based on the culture of the rapidly growing mouse hybridoma cell line 1E6 in a defined serum-free culture medium. The use of serum-free conditions greatly increases the sensitivity to toxic substances, due to the absence of binding proteins. The testing of known toxic agents showed that this assay disclosed cytotoxicity with a high sensitivity. The Hybritest thus provides a simple yet sensitive and reproducible bioassay for quality control of culture media, water, chemical compounds and equipment in an IVF programme. Tests of different batches of culture media showed that media for IVF should be processed from powder and high-quality sterile water. It is important not only to test the single components of the culture media, but also the final product in a sensitive test system.

Effect of a low fat diet on essential fatty acid metabolism in healthy human subjects.

The fatty acid pattern in phospholipids of serum and platelets, and conversion of labelled 18:3(n-3) and 20:3(n-6) in cultured lymphocytes was studied in healthy subjects consuming a low fat diet for four weeks. In serum and platelets the level of polyunsaturated C20 and C22 fatty acids was maintained stable, while the content of 18:2(n-6) was significantly decreased. The delta 6-desaturase activity in lymphocytes was increased after the low fat diet, while the rate of delta 5-desaturation was unchanged. It is concluded that the rate of delta 6-desaturation and perhaps also chain-elongation is regulated by the content and composition of dietary lipids in order to maintain the fatty acid pattern of phospholipids stable.

Disorders related to the metabolism of phytanic acid.

The phytanic acid found in man stems from exogenous sources, mainly as minor parts of fish and animal fats. Free phytol, which is easily converted to phytanic acid in mammals, is present in fats of vegetable origin. Healthy individuals are able to degrade the small amounts of phytanic acid and phytol which are ingested. Accumulation of phytanic acid has been considered diagnostic for Refsum's disease, and a prerequisite for this diagnosis. However, a few patients with proven Refsum's disease have eliminated their phytanic acid stores by dietary means. Two healthy mothers of patients with Refsum's disease have been reported, in whom serum phytanic acid was considerably increased. Furthermore, phytanic acid has recently been found in patients with several socalled peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, hyperpipecolic acidemia, rhizomelic chondrodysplasia punctata, Leber disease). Skin fibroblasts both from patients with classical Refsum's disease and from those with the peroxisomal disorders have a defect in the alpha-oxidation of phytanic acid, with a residual enzyme activity less than 10% of normal. The presence of this defect in the patients with peroxisomal disease makes it tempting to suggest that alpha-oxidation of phytanic acid normally takes place in the peroxisomes. Subcellular studies in rat liver show, however, unequivocally that the alpha-oxidation of phytanic acid is located to the mitochondria. Thus, patients with the peroxisomal syndromes must probably have a defect also in the mitochondria, in addition to the many peroxisomal deficiencies.