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Abstract: α-SNAP is an essential component of the protein machinery responsible for membrane fusion events in different cell types. The hyh (hydrocephalus with hop gait) mouse carries a missense mutation in Napa gene that results in a point mutation (M105I) in α-SNAP protein. Homozygous animals for the mutant allele have been identified by the clinical and/or neuropathological phenotype, or by direct sequencing of PCR products. The aims of the present study were (i) to develop a high-throughput technique to genotype hyh mice, (ii) to correlate genotype-phenotype, and (iii) to analyze the earliest pathological changes of hyh mutant mice. As no restriction sites are affected by the hyh mutation, we resolved this problem by creating a BspHI restriction site with a modified (mismatch) polymerase chain reaction (PCR) primer in wild-type allele. This artificially created restriction site (ACRS)–PCR technique is a simple, rapid and reliable method to genotype hyh mice in a day-work procedure. Biochemical and histological analysis of genotyped hyh embryos at different developmental stages allowed us to identify and characterize the earliest brain pathological changes of the hyh phenotype, including the first signs of neuroepithelial disruption and neuronal ectopia. In addition, genotype-phenotype analysis of 327 animals confirmed that (i) hyh is a single-gene autosomal recessive disorder, and (ii) the disorder has 100% penetrance (i.e., the mutation was only present in affected mice). The genotyping method described here enhances the potentiality of hyh mouse as a unique in vivo model to study the role of membrane trafficking in different developmental and physiological processes. [Copyright &y& Elsevier]

Abstract: The hyh mouse carrying a point mutation in the gene encoding for soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein alpha (α-SNAP) develops inherited hydrocephalus. The investigation was designed to study: (i) the clinical evolution of hyh mice; (ii) factors other than the α-SNAP mutation that may influence the expression of hydrocephalus; (iii) the neuropathological features underlying the different forms of clinical evolution. The study included 3017 mice, 22.4% of which were hydrocephalic. The neuropathological study was performed in 112 mice by use of light and electron microscopy. It was found that maternal- and sex-related factors are involved in the heterogeneous expression of hyh phenotype. The clinical evolution recorded throughout a 4-year period also revealed a heterogeneous expression of the hydrocephalic phenotype. Two subpopulations were distinguished: (i) 70% of mice underwent a rapidly progressive hydrocephalus and died during the first 2 months of life; they presented macrocephaly, extremely large expansion of the ventricles, equilibrium impairment and decreased motor activity. (ii) Mice with slowly progressive hydrocephalus (30%) survived for periods ranging between 2 months and 2 years. They had no or moderate macrocephaly; moderate ventricular dilatation and preserved general motor activity; they all presented spontaneous ventriculostomies communicating the ventricles with the subarachnoid space, indicating that such communications play a key role in the long survival of these mice. The hyh mutant represents an ideal animal model to investigate how do the brain “adapt” to a virtually life-lasting hydrocephalus. [Copyright &y& Elsevier]