Generation and phenotyping of a targeted mouse model of alkaptonuria

Purpose: Alkaptonuria (AKU) is a rare autosomal recessive metabolic disease caused by mutations in the gene homogentisate 1,2-dioxygenase enzyme (HGD). Deficiency of HGD leads to an accumulation of homogentisic acid in the blood and tissues. Overtime, HGA is polymerised to form a pigment which deposits into connective tissues, particularly in cartilage. The cartilage becomes brittle and osteoarthropathy manifests in early adulthood, becoming very debilitating as it progresses. Studying rare diseases with extreme phenotypes like AKU aid our understanding about more common disorders like osteoarthritis. The current mouse model for AKU was generated by chemical mutagenesis. To overcome the uncertainty of this model harbouring other potentially confounding uncharacterised mutations, we generated a specific HGD (Homogentisate 1,2-dioxygenase) null mouse with the advantage of conditional deletion.
Methods: Embryonic stem cells in the C57BL6 background with disrupted HGD gene function were obtained from the Knockout Mouse Project (KOMP). This knockout first plasmid contains the insertion of an IRES:lacZ trapping cassette and a promoter-driven neo cassette into the fifth intron of the HGD gene with the sixth exon flanked by LoxP sequences (HGD tm1a). Flp recombinase reverts the mutation back to wildtype with a floxed critical exon (HGD tm1c). The model then becomes conditional though Cre recombinase which removes the floxed exon resulting in a mutant transcript (HGD tm1d). The Hgd tm1a knock-out first model is characterised here.
Results: Homozygous tm1a mice show black urine stained cage bedding, one of the first AKU symptoms. Homogentisic acid is elevated in the homozygous tm1a in both the urine (99,575 μmol/L) and plasma (100.5 μmol/L) compared to C57BL6 wildtype mice (urine; 0.9 μmol/L, plasma; 1.7 μmol/L). Heterozygous tm1a and tm1c mice have levels of HGA comparable to the C57BL6 wildtype. Ochronosis, pigmentation of chondrocytes found within calcified articular cartilage, was detected at 9 weeks in the tm1a mouse as pericellular pigmentation. At 26 and 40 weeks, numerous fully pigmented chondrocytes can be seen in the homozygous tm1a knee cartilage with no evidence of ochronosis in heterozygous mice. The β-galactosidase transgene (LacZ) that is knocked in to the tm1a mouse to disrupt gene function was used to localise HGD expression. Whole-mount staining of several adult tissues from tm1a mice shows positive expression in the liver and kidney cortex but no other organs in development nor in adult mice. β-galactosidase staining demonstrates transgene activity throughout the parenchymal cells and is confined to the proximal convoluted tubule cells of the kidney cortex.
Conclusions: This novel conditional HGD null mouse exhibits all the main symptoms of AKU. There is no evidence of haploinsufficiency. The knock in LacZ transgene was successfully used to localise HGD expression.