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

Constraint-Based Infinite Model Checking and Tabulation for Stratified CLP

Forward analysis procedures for infinite-state systems such as timed systems were limited to safety properties. We give the first constraint-based forward analysis for infinite-state systems that goes beyond safety properties. Namely, we take the restriction of the $\mu$-calculus to least-fixpoint formulas where negation is applied to closed subformulas only. We characterize these properties as perfect models of constraint logic programs, and we present a tabulation procedure for the top-down evaluation of stratified constraint logic programs

<i>aldh7a1</i> Regulates Eye and Limb Development in Zebrafish

<div><p>Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during development. Although mutations in numerous genes have been described, these account for a minority of cases, complicating molecular diagnosis and genetic counseling. Here we describe a key role of <i>aldh7a1</i> as a gene necessary for normal eye development. We show that morpholino knockdown of <i>aldh7a1</i> in zebrafish causes uveal coloboma and misregulation of <i>nlz1</i>, another known contributor to the coloboma phenotype, as well as skeletal abnormalities. Knockdown of <i>aldh7a1</i> leads to reduced cell proliferation in the optic cup of zebrafish, delaying the approximation of the edges of the optic fissure. The <i>aldh7a1</i> morphant phenotype is partially rescued by co-injection of <i>nlz1</i> mRNA suggesting that <i>nlz1</i> is functionally downstream of <i>aldh7a1</i> in regulating cell proliferation in the optic cup. These results support a role of <i>aldh7a1</i> in ocular development and skeletal abnormalities in zebrafish.</p></div

Clinical diagnosis of presumed SOX2 gonadosomatic mosaicism.

PURPOSE: To describe a family with presumed SOX2 gonadosomatic mosaicism diagnosed upon ophthalmic examination of the proband’s mother. METHODS: The family underwent comprehensive ophthalmic and physical examination. Variant detection was performed using trio exome analysis on peripheral leukocyte DNA from blood and saliva samples. Variant segregation analysis was performed using a custom panel NGS sequencing. An identified variant in the SOX2 gene was confirmed in the proband by Sanger sequencing. RESULTS: We report an individual with bilateral microphthalmia, developmental delay, hearing loss, and dysmorphic features. Her mother was found to have asymptomatic forme fruste uveal coloboma affecting her anterior segment. Her father, aunt, and sisters were unaffected. Trio exome sequence analysis showed an apparent de novo heterozygous deletion in the proband, NM_003106.3:c.70_89del, NP_003097.1:p. (Asn24Argfs*65), classified as pathogenic. Testing of the other family members’ peripheral blood and saliva was negative for this variant. The iris transillumination abnormalities in the proband’s mother supports a gonadosomatic mosaicism scenario. CONCLUSIONS: The results from this family underscore the importance of performing detailed evaluations of the parents of apparently sporadically affected individuals with heritable ophthalmic disorders. The identification of mildly affected individuals could substantially alter recurrence risks

Expression pattern of <i>aldh7a1</i> in zebrafish.

<p>Whole-mount <i>in situ</i> hybridization of <i>aldh7a1</i> at (A) 24 hpf and (B) 48 hpf. L, lens; OF, optic fissure; PF, pectoral fin. Scale bar: 65 µm in A; 60 µm in B.</p

Aldh7a1 is required for retinal cell proliferation.

<p>(A–B) Dividing cells in developing eye were labeled with phosophohistone-3 antibody (H3P) in (A) Control MO and (B) <i>alh7a1</i>morphant embryos at 28 hpf. (A) and (B) are projection images of z-stacks through the depth of the eye. (C) Average number of dividing cells per eye quantified for control MO (n = 6), Nlz1 MO (n = 7), <i>nlz1</i> mRNA (n = 6) rescued Statistical significance indicated above columns *P<0.05, **P<0.01, ***P<0.0001. Scale bar: 65 µm.</p

Expression pattern of genetic eye development markers in control and <i>aldh7a1</i> morphant embryos.

<p>(A) Expression of <i>nlz1</i> in optic fissure is down-regulated in (B) <i>aldh7a1</i> morphant fish. <i>vax2</i> and <i>pax2.1</i> do not seem to show significant change in expression between control MO (C,E) and <i>nlz1</i> morphant (D, F) fish. (G) Co-injection of <i>nlz1</i> mRNA resulted in partial rescue of <i>aldh7a1</i> MO phenotype, examined at 28 hpf, while co-injection of <i>vax2</i> mRNA showed no change. Scale bar: 65 µm.</p