In Vivo Suppressor Mutations Correct a Murine Model of Hereditary Tyrosinemia Type I

In vivo suppressor mutations correct a murine model of hereditary tyrosinemia type I Kara Manning*†, Muhsen Al-Dhalimy*, Milton Finegold‡, and Markus Grompe*§ Departments of *Molecular and Medical Genetics and §Pediatrics, Oregon Health Sciences University, Portland, OR 97201; and ‡Department of Pathology, Texas Children’s Hospital, Houston, TX 77030 Edited by Leon E. Rosenberg, Princeton University, Princeton, NJ, and approved July 29, 1999 (received for review June 9, 1999) Hereditary tyrosinemia type I and alkaptonuria are disorders of of two shorter transcripts that predict severely truncated HGD tyrosine catabolism caused by deficiency of fumarylacetoacetate proteins (10). These mice have high levels of homogentisic acid in hydrolase (FAH) and homogentisic acid dioxygenase (HGD), respec- the urine; however, older mice show no evidence of ochronosis or tively. Tyrosinemia is a severe childhood disease that affects the liver joint disease, possibly because mice can synthesize ascorbic acid, and kidneys, but alkaptonuria is a more benign adult disorder in unlike humans (9). A knock-out mouse model of HT1 also exists comparison. Because HGD is upstream of FAH in the tyrosine path- (FAH⌬exon5) (11), which is lethal unless kept on NTBC (12). When way, mice doubly mutant in both enzymes were found to be pro- taken off NTBC, FAH⌬exon5 mice experience liver failure, showing tected from the liver and renal damage of tyrosinemia as hypothe- necrosis, inflammation, and dysplastic cells histologically. They also sized. Mice mutant at the tyrosinemic locus but heterozygous for exhibit renal tubular disease and will develop hepatic cancer even alkaptonuria spontaneously developed clonal nodules of functionally while on NTBC (12). By breeding these two mouse models, we normal hepatocytes that were able to rescue the livers of some mice tested the hypothesis that a block in the pathway at HGD could with this genotype. This phenotypic rescue was a result of an rescue FAH knock-out mice from the severe HT1 phenotype. inactivating mutation of the wild-type homogentisic acid dioxygenase gene, thus presenting an example of an in vivo suppressor Materials and Methods mutation in a mammalian model. Strains of Mice and Animal Husbandry. Aku mice were obtained from Xavier Montagutelli at the Pasteur Institute (Paris). Aku and ⌬exon5 ereditary tyrosinemia type I (HT1) is an inborn error of Fah mice (11) were bred to create double heterozygotes, Hmetabolism caused by deficiency of fumarylacetoacetate hy- which then were crossed to obtain the desired genotypes (without drolase (FAH), the enzyme that carries out the last step of the controlling for strain background). Pups were genotyped at the Fah tyrosine catabolic pathway (Fig. 1) (1, 2). Of the four genetic locus by using a 3-primer PCR on 200 ng of tail-cut DNA as diseases known to result from deficiencies in specific enzymes in the described (11). Aku mice were genotyped as described by PCR (10). pathway, HT1 is the most severe and is characterized by progressive All breeders and all mutant animals were treated with NTBC ͞ liver dysfunction and renal tubular disease (2). Moreover, HT1 is containing drinking water at a concentration of 7.6 mg liter (pro- unique among the tyrosine catabolic pathway diseases in that vided by S. Lindstedt, Gottenburg, Sweden). FAH mutants with patients have a high risk for developing liver cancer (3). Maleyla- varying Hgd genotypes were kept on NTBC until an age of at least cetoacetate (MAA) and fumarylacetoacetate (FAA), the two 2 months, when NTBC was removed. Weights were followed at a compounds that precede FAH in the pathway and, thus, accumu- weekly basis, and animals were sacrificed at 4 weeks after stopping late in FAH-deficient hepatocytes are predicted to be alkylating NTBC or at a later time point for those that survived NTBC agents by structure and are the primary cause of the liver damage withdrawal for 7 or more weeks. Animal care and experiments were (1, 4). Currently, the drug NTBC (2-(2-nitro-4-trifluoro- all in accordance with the Guidelines of the Department of Animal methylbenzoyl)-1,3-cyclohexanedione), which blocks the pathway Care at Oregon Health Sciences University. upstream of the formation of either MAA or FAA by inhibiting 4-hydroxyphenylpyruvate dioxygenase (HPD) (Fig. 1), is used to HGD Enzyme Assay. Liver was homogenized and then sonicated for ϫ ␮ treat patients with HT1 (5). However, NTBC has the disadvantage 2 10 sec in 500 l of 0.25 M sucrose. The liver extract then was of markedly elevating blood tyrosine levels and, therefore, needs to clarified by centrifugation (full speed in a microcentrifuge for 15 ␮ be combined with dietary restriction of phenylalanine and tyrosine, min at 4°C), and 50–200 g of protein was added to the reaction mixture containing 100 mM potassium phosphate buffer, pH 7͞2 because increased tyrosine may lead to corneal ulcers and neuro- ͞ ␮ ͞ ␮ logical problems as is seen in patients with hereditary tyrosinemia mM ascorbate 50 M iron sulfate 250 M homogentisate. The types II and III (Fig. 1) (2). Additionally, NTBC may not prevent production of maleylacetoacetate was measured spectrophoto- hepatocarcinomas in patients with HT1 (3, 6). metrically at 330 nm, as described (13, 14). Deficiency of another enzyme upstream of FAH in the pathway, Biochemical Analysis and Quantitative Amino Acid Analysis. Individ- homogentisate 1,2 dioxygenase (HGD), causes alkaptonuria (aku), ␮ which has no increase in tyrosine levels (7). Instead, there is an ual plasma samples of 25 l were analyzed on a Beckman 6300 accumulation of the substrate for HGD, homogentisic acid, which automated amino acid analyzer (15). Samples from animals were is excreted in the urine and can be oxidized spontaneously to a black pigment, causing darkly colored urine (8). This pigment also is This paper was submitted directly (Track II) to the PNAS office. deposited in many tissues, causing ochronosis, arthritis, and con- Abbreviations: HT1, hereditary tyrosinemia type I; aku, alkaptonuria; FAH, fumarylaceto- nective tissue damage in adulthood (7). acetate hydrolase; HGD, homogentisic acid dioxygenase; MAA, maleylacetoacetate; FAA, Because elevated blood tyrosine and liver cancer are not found fumarylacetoacetate; NTBC, 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione; in aku patients, and the toxic compounds MAA and FAA do not HPD, 4-hydroxyphenylpyruvate dioxygenase; RT-PCR, reverse transcription–PCR; H&E, he- accumulate, we hypothesized that inhibition of the pathway at HGD matoxylin͞eosin; ASO, allele-specific oligonucleotide. may be a better treatment for HT1 patients than blocking at HPD †To whom reprint requests should be addressed at: Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Way, L103, Portland, OR 97201. E-mail: manningk@ by using NTBC. This hypothesis could be tested by using the mouse ohsu.edu. models of aku and HT1. An aku mouse model has been described The publication costs of this article were defrayed in part by page charge payment. This that was created by N-ethyl-N-nitrosourea mutagenesis (9). Aku article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. mice were found to have a splice mutation, causing the production §1734 solely to indicate this fact. 11928–11933 ͉ PNAS ͉ October 12, 1999 ͉ vol. 96 ͉ no. 21 Downloaded by guest on September 30, 2021 sition 866 had the sequence 5Ј-TAATACGACTCACTATAGG- GGCCTGGTATGAAGATCG-3Ј. Cloning and Sequencing. The PCR products derived from the 3Ј half of Hgd were separated by electrophoresis to isolate the wild-type- length product (see Fig. 4). This fragment was isolated by using a QIAquick gel extraction kit (Qiagen) and then cloned with either AdvanTAge PCR cloning kit (CLONTECH) or a PCR-Script kit for Pfu products (Stratagene). PCR was performed on the resulting bacterial colonies by using the same PCR protocol as described above, except only 25 cycles were used. Dideoxy sequencing (18) by using the same forward primer as listed above then was performed on the PCR products after cleaning with a QIAquick PCR purifi- cation kit (Qiagen). Allele-Specific Oligonucleotide (ASO) Hybridization. Fourteen- to 15-bp oligonucleotides homologous to the wild-type and various mutant sequences were designed (19). Ten microliters of the PCR product from PCR-2 clones (from colonies, as described above) was denatured in 100 ␮l 0.4 M NaOH͞25 mM EDTA and spotted onto a Hybond-Nϩ membrane (Amersham) in a Schleicher & Schuell Fig. 1. Tyrosine-degradation pathway and its diseases. dot-blot apparatus. Five picomoles of each ASO was end-labeled by T4 polynucleotide kinase and [␥-32P]ATP. The kinase was heat- obtained as follows. Animals were killed by decapitation and blood inactivated, and 50 pmol of the opposing, unlabeled ASO was added. The dot blots were hybridized overnight at 25–45°C (de- was collected. For anticoagulation, the blood was mixed immedi- pending on the calculated T )in6ϫ SSPE͞1% SDS͞5ϫ Den- ately with 10 ␮l of sodium-heparin. The red blood cells were m hardt’s solution. Washes were carried out for 10–30 min with either removed by a brief centrifugation, and the plasma was frozen at 5ϫ or 2ϫ SSC and 0.5% SDS at 25–42°C. Ϫ80°C. Twenty microliters of plasma were mixed with 80 ␮lofa solution of 7% BSA and assayed for alanine aminotransferase, Restriction Enzyme Mutation Analysis. Three mutations were con- bilirubin, and creatinine levels with a Kodak Ektachem 700 chem- firmed by altering a base pair in a PCR primer near the mutation istry analyzer. to create a restriction site. For mutation A 3 G at 998, primer 5Ј-ATGGAAACTACACACCCTTC-3Ј created a TaqI site recog- Histology and Immune Histology. For immunohistology, tissues were nizing the mutant sequence. For mutation A 3 C at 1154, primer fixed in 10% phosphate-buffered formalin, pH 7.4, dehydrated in 5Ј-GTAATAAGGAGGTCTGAACG-3Ј created a Psp 14061 100% ethanol, and embedded in paraffin wax at 58°C.

Load more