Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human

Papillorenal syndrome (PRS, also known as renal-coloboma syndrome) is an autosomal dominant disease characterized by potentially-blinding congenital optic nerve excavation and congenital kidney abnormalities. Many patients with PRS have mutations in the paired box transcription factor gene, PAX2. Although most mutations in PAX2 are predicted to result in complete loss of one allele's function, three missense mutations have been reported, raising the possibility that more subtle alterations in PAX2 function may be disease-causing. To date, the molecular behaviors of these mutations have not been explored. We describe a novel mouse model of PRS due to a missense mutation in a highly-conserved threonine residue in the paired domain of Pax2 (p.T74A) that recapitulates the ocular and kidney findings of patients. This mutation is in the Pax2 paired domain at the same location as two human missense mutations. We show that all three missense mutations disrupt potentially critical hydrogen bonds in atomic models and result in reduced Pax2 transactivation, but do not affect nuclear localization, steady state mRNA levels, or the ability of Pax2 to bind its DNA consensus sequence. Moreover, these mutations show reduced steady-state levels of Pax2 protein in vitro and (for p.T74A) in vivo, likely by reducing protein stability. These results suggest that hypomorphic alleles of PAX2/Pax2 can lead to significant disease in humans and mice

Pax2 immunofluoresence on COS-7 cells transfected with wild-type or mutant <i>Pax2</i> expression vectors.

<p>Both wild-type and mutant proteins display nuclear localization, as evidenced by co-localization of green fluorescence (Pax2) with DAPI nuclear staining (blue).</p

Clinical ocular phenotype in C57BL/6-<i>Pax2^+/A220G</i> mice compared to wild-type, C57BL/6 mice.

<p>(A) Fundus photograph of C57BL/6 mouse showing normal optic nerve and radial pattern of retinal blood vessels. (B) Fundus photograph of C57BL/6-<i>Pax2^+/A220G</i> mouse showing congenital excavation of the optic nerve head with peripapillary pigment changes (arrow). (C) Lectin immunofluorescence of wild-type C57BL/6 mouse showing normal, radial vessel patterning. (D,E) Lectin immunofluorescence of C57BL/6-<i>Pax2^+/A220G</i> mice showing abnormal vascular patterning, including curving of vessels towards the dorsal retina (D, arrows, d = dorsal, v = ventral) and separation of the central retinal vascular trunks (E, arrows). Histologic section of a <i>Pax2^+/+</i> (F) and a <i>Pax2^+/A220G</i> (G) mouse eye through the optic nerve and peripapillary retina showing abnormal excavation of the optic nerve (G, arrow) and retinal rosette formation (G, arrowhead). Remnants of the <i>tunica vasculosis lentis</i> and mild extension of the retinal pigment epithelium were variably noted in histopathology from other <i>Pax2^A220G/+</i> eyes (data not shown).</p

Histologic sections of <i>Pax2^+/+</i> and <i>Pax2^A220G/A220G</i> mouse kidneys (axial) and cerebellum (sagittal) at E17.5.

<p>Whereas wild-type mice have begun to develop renal glomeruli (arrow, A) and tubules (arrowhead, A), the mutant mice have only primordial kidneys with poor differentiation of these structures (arrow, B) In contrast, the differentiation of the cerebellum of both wild-type (C) and mutant (D) mice is comparable at this time, despite the midbrain-hindbrain boundary being a site of <i>Pax2</i> expression during embryogenesis. By E14.5, cranial structure was grossly normal in both wild-type (E) and homozygous mutant (F) embryos.</p

Electrophoretic mobility shift assay comparing DNA binding of wild-type and three mutant Pax2 proteins.

<p>A labeled Pax2 DNA-binding consensus sequence was incubated in the presence or absence of nuclear extract of COS-7 cells expressing equal amounts of the wild-type or mutant Pax2 protein; the same, unlabeled, competing DNA oligonucleotide; and/or a mutated version of the unlabeled oligonucleotide (Mut-Pax2). Nuclear extracts from mock transfected cells did not appreciably result in a shift of the labeled Pax2 DNA-binding site oligonucleotide, whereas wild-type and all three mutant Pax2 proteins bound the oligonucleotide with approximately equal affinity. Specificity for this binding was shown by competing the binding with the same, unlabeled oligonucleotide sequence and by failure of an unlabeled mutant oligonucleotide to compete for binding.</p

Comparison of <i>Pax2</i> mRNA steady-state levels and Pax2 protein stability in wild-type and mutant expression vector-transfected NIH/3T3 cells.

<p>Although steady-state levels of <i>Pax2</i> mRNA were comparable in wild-type and mutant transfected cells (A), the short-term protein stability of mutant Pax2 protein products were considerably reduced compared to wild-type, as determined in cycloheximide translation-inhibition experiments (B,C). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000870#pgen-1000870-t001" target="_blank">Table 1</a> for quantification of mRNA levels.</p

Threshold cycle (C_t) quantification of real-time, reverse-transcriptase PCR of wild-type and mutant <i>Pax2</i> transfected NIH/3T3 cells shows no significant difference in steady-state levels of <i>Pax2</i> mRNA relative to Gapdh.

<p>Threshold cycle (C_t) quantification of real-time, reverse-transcriptase PCR of wild-type and mutant <i>Pax2</i> transfected NIH/3T3 cells shows no significant difference in steady-state levels of <i>Pax2</i> mRNA relative to Gapdh.</p

Comparison of wild-type and mutant Pax2 protein transactivation and expression in cell culture.

<p>NIH/3T3 cells were transfected with expression constructs for wild-type or mutant <i>Pax2</i> along with a <i>Pax2</i>-responsive luciferase reporter gene. All three mutants tested show reduced ability to transactivate (A). When steady-state levels of Pax2 protein were compared on Western blots from these experiments, mutants showed consistently lower levels of expression (B). Similar findings were observed when these experiments were replicated in COS-7 cells (data not shown).</p

Homology modeling of the wild-type and mutant Pax2 paired domain-DNA complex.

<p>The paired domain of wild-type Pax2 domain DNA are represented by red and white ribbons, respectively, and their corresponding atomic structures are shown by red and white bonds (A). Hydrogen bonds are shown in blue. Threonine 74 in the mouse protein sequence (equivalent to T75A in human) is absolutely conserved across several species (B) and across all known murine Pax-family members (C). Fragments of the Pax2 paired domain–DNA complex modified by the mutations T74A, dup73ET and G75S are shown on (D–F), respectively. Hydrogen bonds presented in the wild type protein that are broken by the mutation T74A are labeled as 1 and 2 for (D) and by the mutation G75S is labeled as 3 (F). Yellow arrows indicate the location of mutations in Pax2 paired domain. A schematic of the Pax2b protein modified from Lechner <i>et al. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000870#pgen.1000870-Lechner1" target="_blank">[32]</a> showing the paired domain (gray), the octapeptide (Oct) domain (yellow), and the C-terminus, which is rich in proline, serine, threonine and tyrosine (PSTY) residues (G). Numbers indicate amino acid position. The arrow denotes the approximate position of the three mutations studied.</p

Nitisinone improves eye and skin pigmentation defects in a mouse model of oculocutaneous albinism

Mutation of the tyrosinase gene (TYR) causes oculocutaneous albinism, type 1 (OCA1), a condition characterized by reduced skin and eye melanin pigmentation and by vision loss. The retinal pigment epithelium influences postnatal visual development. Therefore, increasing ocular pigmentation in patients with OCA1 might enhance visual function. There are 2 forms of OCA1, OCA-1A and OCA-1B. Individuals with the former lack functional tyrosinase and therefore lack melanin, while individuals with the latter produce some melanin. We hypothesized that increasing plasma tyrosine concentrations using nitisinone, an FDA-approved inhibitor of tyrosine degradation, could stabilize tyrosinase and improve pigmentation in individuals with OCA1. Here, we tested this hypothesis in mice homozygous for either the Tyrc-2J null allele or the Tyrc-h allele, which model OCA-1A and OCA-1B, respectively. Only nitisinone-treated Tyrc-h/c-h mice manifested increased pigmentation in their fur and irides and had more pigmented melanosomes. High levels of tyrosine improved the stability and enzymatic function of the Tyrc-h protein and also increased overall melanin levels in melanocytes from a human with OCA-1B. These results suggest that the use of nitisinone in OCA-1B patients could improve their pigmentation and potentially ameliorate vision loss