A missense mutation (Q279R) in the fumarylacetoacetate hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation

Abstract

Background: Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules.

Results: Analysis of DNA from a FAH expressing region showed that the expression of the protein was due to correction of the Q279R mutation. RT-PCR was used to assess if Q279R RNA was produced in the liver cells and in fibroblasts from the patient. Normal mRNA was found in the liver region where the mutation had reverted while splicing intermediates were found in non-expressing regions suggesting that the Q279R mutation acted as a splicing mutation in vivo. Sequence of transcripts showed skipping of exon 8 alone or together with exon 9. Using minigenes in transfection assays, the Q279R mutation was shown to induce skipping of exon 9 when placed in a constitutive splicing environment.

Conclusion: These data suggest that the putative missense mutation Q279R in the FAH gene acts as a splicing mutation in vivo. Moreover FAH expression can be partially restored in certain liver cells as a result of a reversion of the Q279R mutation and expansion of the corrected cells.

Figure 1

Mutation analysis in different liver regions. DNA was extracted from different liver regions and amplified by PCR. PCR products were digested with either Alu I to detect IVS6-1g->t or with Msp I to detect Q279R. For IVS6-1 g->t, the same heterozygous pattern is seen in both the reverted nodule (NT), tumor section (T) and fibroblast DNA (F), showing 3 bands, one at 156-, another at 104- and the last at 75-bp. The control (wt/wt) shows two bands, one at 156- and the other at 75-bp, indicating the absence of IVS6-1g->t (M: molecular weight marker, 100- and 200-bp). For Q279R both the 78- and 58-bp bands are seen in the tumor section (T) and fibroblast DNA (F) indicating an heterozygous genotype while only the 78-bp wild-type band is seen in the control (wt/wt). In the region suspected of reversion (NT), a strong 78-bp wild-type band is seen with a weak 58-bp mutated band (M: molecular weight marker, 100-bp).

Figure 2

RT-PCR on RNA from different liver sections and fibroblasts. Total RNA was extracted from various samples, reverse transcribed and amplified by PCR. The 537-bp fragment starts in the middle of exon 6 and ends in the middle of exon 12. A- RT-PCR products amplified from total RNA extracted from different liver regions i.e. non-tumoral (lane 2) and tumoral (lane 3) and from fibroblasts of the patient (lane 4). (lane 1: Control RNA from T19-EBV normal cells). B- RT-PCR products amplified from total RNA extracted from transformed lymphocytes of the parents (father; lane 1, mother; lane 2) and cultured fibroblasts of another patient homozygous for IVS6-1g->t (lane 3). A normal length cDNA is seen in the reverted nodule (537-bp, A, lane 2) and in both parents. Three of the products seen in the tumor liver section (A' lane 3) are also observed in a patient homozygous for the IVS6-1g->t mutation (B, lane 3).

Figure 3

Diagram of RNA alternative transcripts found in the IVS6-1g->t and Q279R patient.The splicing pattern is based on sequence analysis of the RT-PCR transcription products amplified with the primers Tan5 and Tan 51. The alternative transcripts identified are pictured from exons 6 to 10 (represented by boxes). The introns are represented by lines. Black boxes indicate that a cryptic splice site is activated with a subsequent skipping of exonic sequences. Premature stop codons that appear due to frameshift are represented by a stop sign.

Figure 4

Analysis of the splicing pattern obtained with the minigenes. A- The splicing K7 consists of exon 1 of β-globin and its downstream intronic sequences joined to β-globin exon 3 and its upstream intronic sequences. Exon 9, with or without the Q279R mutation was inserted in K7 at the intronic junction. HeLa cells were transiently transfected with both constructs, the wild-type Q279Q-K7 and Q279R-containing Q279R-K7. After 24 hours, cells were harvested and the splicing pattern of each minigene was examined by RT-PCR analysis of the transcripts. Exons are represented by boxes and introns by lines. The primers used for RT-PCR are indicated at each end of the splicing K7. B- Total RNA extracted from transfected HeLa cells was amplified with HG1S and HG3AS. Plasmidic DNA Q279Q-K7 and Q279R-K7 (pDNA) were also amplified as a control. The band obtained for Q279Q-K7 transfected cells (RT+) is of expected size, in contrast to the band obtained in the case of Q279R-K7 (RT+) transfected cells, which is of lower molecular weight. RT^- serves as a negative control: the reverse transcription reaction was performed without any enzyme. In the two RT^- fractions, the amplification of about 900-bp is due to plasmidic DNA contamination.