A simple PCR-based genotyping method for M105I mutation of alpha-SNAP enhances the study of early pathological changes in hyh phenotype

Luis Federico Bátiz*, Ruth Roales-Buján, Luis M. Rodríguez-Pérez, Isabel M. Matas, Patricia Páez, María Roque, Antonio J. Jiménez, Cayo Ramos, José Manuel Pérez-Fígares

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


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.

Original languageEnglish
Pages (from-to)281-290
Number of pages10
JournalMolecular and Cellular Probes
Issue number6
StatePublished - Dec 2009
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by grants from FIS, PI 060243, Instituto de Salud Carlos III and Servicio Andaluz de Salud, Spain to JMP-F; FONDECYT 1070241 (Dr. Esteban M. Rodriguez, Universidad Austral de Chile), DID 2005–12 (Universidad Austral de Chile) and CONICYT (Doctoral Fellowship) to LFB. We thank Mr. Genaro Alvial for his excellent technical support. We also thank Dr. Gudrum Kausel, Dr. Jaime Figueroa and Dr. Hans Richter (Universidad Austral de Chile, Valdivia, Chile) and Dr. Juan Young (Centro de Estudios Científicos [CECS], Valdivia, Chile) for their technical suggestions and assistance. We are very grateful to Dr. Esteban M Rodríguez for his support and discussions during the development of this work.


  • Genotyping
  • Napa
  • SNAREs
  • alpha-SNAP
  • hyh mouse


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