Type 1 hereditary tyrosinemia. Evidence for molecular heterogeneity and identification of a causal mutation in a French Canadian patient.

Abstract

IntroductionType 1 hereditary tyrosinemia (HT1) is a metabolic disorder caused by a deficiency of fumarylacetoacetate hydrolase (FAH). Using a full-length FAH cDNA and specific antibodies, we investigated liver specimens from seven unrelated HT1 patients (six of French Canadian and one of Scandinavian origin). The expression of FAH in livers of these individuals was analyzed at several molecular levels including mRNA, immunoreactive material (IRM), and enzymatic activity. Four phenotypic variants were differentiated by these assays: (i) presence of FAH mRNA without any IRM or enzymatic activity, (ii) decreased FAH mRNA, IRM, and enzymatic activity, (iii) moderately decreased FAH mRNA and IRM with severely reduced enzymatic activity, and (iv) undetectable FAH mRNA, IRM, and enzymatic activity. These various molecular phenotypes suggest that this disorder may be caused by a variety of FAH mutations. Interestingly, we found no apparent relationship between the clinical and the molecular phenotypes, except that patients with absent IRM and enzymatic activity tend to have higher levels of serum alpha-fetoprotein and an earlier clinical onset. To further analyze the molecular basis of HT1, the FAH cDNA of a patient designated as variant A was amplified and sequenced. An A-to-T transversion, which substitutes asparagine'6 with isoleucine (N161), was identified. This patient was heterozygous as shown by direct sequencing of the amplified region and hybridization with allele-specific oligonucleotide probes. The N161 allele originates from the father and the second allele appears not to be expressed in the liver of the proband. CV-1 cells transfected with the mutant cDNA produced FAH mRNA, but no protein or hydrolytic activity, as predicted by the "A" phenotype of the patient. This is the first demonstration of heterogeneity in the expression of FAH at the levels of protein, mRNA, and enzymatic activity in the livers of HT1 patients and is the first identification of a causal mutationin this disease.

Complete Rescue of Lethal Albino c Mice by Null Mutation of 4-Hydroxyphenylpyruvate Dioxygenase and Induction of Apoptosis of Hepatocytes in These Mice by in VivoRetrieval of the Tyrosine Catabolic Pathway

“…Sudden apoptotic death of unmasked phenotype of HT1 in mature and unmodified hepatocytes had not been expected (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)16), and these are implications for the pathogenesis and treatment of liver disease in HT1 patients. We suggest that mature and unmodified hepatocytes in those with the FAH defect cannot survive and that hepatocytes in the chronic form of HT1 have to be protected from a likely acute death.…”

“…The cause of HT1 is mutations in the fumarylacetoacetate hydrolase gene Fah, encoding the last enzyme in the tyrosine catabolic pathway (1,3). In the acute form of the disease, the liver is sometimes nonfunctional by the time the infant is 2-3 months old (1,2).…”

Rescue of mice homozygous for lethal albino deletions: implications for an animal model for the human liver disease tyrosinemia type 1.

“…HT1 is a liver disease of varying severity and clinical outlook (Kvittingen 1986;Goldsmith and Laberge 1989). The severity may be related to the degree of Fah deficiency, the molecular basis of which is heterogeneous (Tanguay et al 1990;Phaneuf et al 1992). The mouse and human phenotypes differ strikingly, however, in that lethal albino mice die within hours after birth, whereas absolute Fah deficiency in humans is tolerated for many months, albeit with severe liver failure.…”

“…Less than 4% of wild-type levels of Fah enable the majority of genotypically rescued mice to survive the critical newborn period and are sufficient for viability and the restoration of a near normal pattem of gene expression. In humans, deficiency of Fah to -30% of normal leads to a chronic liver disease with multiple histological changes and a high predisposition to childhood hepatoma (Dehner et al 1989;Russo and O'Regan 1990;Tanguay et al 1990;Phaneuf et al 1992). In the limited histological analyses carried out to date, rescued mice of line 921 show no consistent liver pathology (G. Kelsey, unpubl.).…”

“…For immunoblots, duplicates of liver homogenates (20--40 ~g) were electrophoresed, transferred, and immunoblotted with an affinity-purified rabbit anti-rat Fah antibody and a secondary 125I-labeled goat anti-rabbit immunoglobulin G. Fah was quantified by scanning the autoradiographs and counting the radioactivity in the immunoreactive band {Tanguay et Phaneuf et al 1992). …”