Box 3 Animal models

First attempts to study the Adnp gene function was initiation by generation of a complete knockout mouse model by incorporation of a neomycin cassette in exons 3–5 in a 129/Sv-derived embryonic stem cells, crossed with C57BL/6 mice and propagated on a mixed genetic background [11]. Full knockouts, lacking the entire protein coding sequence of the Adnp gene, are embryonically lethal and die around E8.5–9.0 with failure of the neural tube closure, suggesting a role in brain development. For this reason, heterozygous male-mutant mice were created which are haploinsufficient for one of the two Adnp alleles. These mice showed the expected 50% reduction in Adnp RNA and protein levels and exhibited cognitive deficits in the Morris water maze in adolescence and older age [13]. No obvious phenotypic abnormalities were mentioned [13], be it that in follow-up studies a small reduction in body size of the mutant animals was measured [122]. However, in the brain, an increase neuronal degeneration markers, some of which reminiscent of tauopathy, was noted. The heterozygous phenotype exhibited a distinctive pattern of abnormally regulated genes. Most notably, Affymetrix array-based expression profiling demonstrated upregulation of a gene family encoding for proteins enriched in the visceral endoderm such as apolipoproteins, cathepsins, and metallothioneins, and downregulation of organogenesis markers including neurogenesis and heart development [63]. When both sexes were analyzed in subsequent experiments, it is striking that male heterozygous knockout mice exhibited deficiencies in an object recognition and social memory test, whereas females were at least partially spared [11]. It should be noted here, though, that in all latter experiments due to a reduced reproductively of the Adnp colony in a mixed genetic background, breeding was continued in ICR outbred mice. Using manganese-enhanced magnetic resonance imaging, axonal transport abnormalities were reported in haploinsufficient mice of both sexes, but transport was reported faster in females than in males [98]. Furthermore, haploinsufficient mice showed reduced dendritic spine density, vocalization impediments, gait, and motor dysfunctions [122]

While it is tempting to draw parallels between the clinical presentation of patients and the abnormalities observed in the mouse model, it should be stressed that the model described is a full deletion and the mutational mechanism of the Helsmoortel–Van der Aa syndrome has not been fully established. For this reason, a mouse model mimicking the most frequent human p.Tyr719* mutation was generated. CRISPR/Cas technology introduced a stop codon at the equivalent position in the mouse genome [52]. The p.Tyr718* mutation is surrounded by a minimal non-coding sequence alterations to facilitate the genome editing process. Heterozygous Tyr mice in a congenic C57BL6/N background express the Tyr mutated allele and the wild-type allele at 50% reduction each on the mRNA level. A truncated protein product in the mutant animals was predicted but not visualized. Phenotypically, Tyr mice had delayed development and with sex-dependent gait defect and syntax abnormalities. Grooming duration and nociception threshold autistic traits were significantly affected in males. Anatomically, dendritic spine densities were reduced an morphologies altered. Early-onset tauopathy in hippocampus and visual cortex was accentuated in males and was paralleled by impaired visual evoked potentials. Expression profiling highlighted among many other dysregulated genes, the decrease in expression of the Foxo3 gene, involved in autophagy

Most recently, we generated a humanized model for the Helsmoortel–Van der Aa syndrome by CRISP/Cas endonuclease mediated genome editing to delete a 14 nucleotide sequence introducing a frameshift mutation just distal to the NES. Mice carrying the heterozygous frameshift p.Leu822Hisfs*6 mutation are viable and fertile and initial investigations show it may become a valid model to study the human condition (D’Incal, Vanden Berghe and Kooy et al., manuscript in preparation). The model is available from the Jackson laboratories as stock 033,128