Elimination of INPP4A and SLC5A7 as candidate genes for congenital progressive ataxia and spastic paresis in pigs

Background: The gene causing congenital progressive ataxia and spastic paresis (CPA) in Large White piglets remains unknown. This lethal neuropathy manifests shortly after birth, and is inherited as a single autosomal recessive allele cosegregating with the microsatellite SW902¹ on SSC3, which approximately corresponds to position 90–110 Mb on HSA2.²INPP4A (inositol polyphosphate-4-phosphatase, type 1) and SLC5A7 (solute carrier family 5, choline transporter, member 7) are attractive positional and functional candidates as they map within this region and are also involved in diseases with similar phenotypes.

A 1-bp deletion in INPP4A causes the weeble (wbl) mutation in mice, a disorder characterized by severe locomotor instability and ataxia.³SLC5A7 encodes a transmembrane transporter in neurons; a missense mutation in a gene of the same family in cattle (SLC35A3) has been shown to cause complex vertebral malformations and locomotor instability.⁴

Radiation hybrid mapping and chromosomal assignments: Porcine INPP4A and SLC5A7 were mapped as previously described,⁵ with the INRA-University of Minnesota porcine radiation hybrid panel IMpRH;⁶ specific experimental conditions are shown in Table S1.

Map assignments were performed with the imprh mapping tool (http://imprh.toulouse.inra.fr/) and are presented in Table 1. SLC5A7 mapped between SW2618 and SWR2069, outside the CPA interval, and was therefore excluded as candidate gene. INPP4A was localized in the CPA region between SW902 and S0094; therefore, it was closely examined for mutations related to the disease.

Sequencing and expression analysis of INPP4A: Total RNA was isolated from 0.5 g of quadriceps muscle using the RNeasy Maxi Kit (Qiagen), and reverse-transcribed with the Superscript II RT-PCR System (Invitrogen Life Technologies).

The entire 2802-bp open reading frame of porcine INPP4A encoded a 105.2-kDa protein of 933 amino acids (http://ca.expasy.org/tools/dna.html) and had high blast sequence homology with horse (92%), human (91%) and mouse (88%) sequences. Comparisons between three normal and three affected piglets revealed only two synonymous SNPs (EU753242: c.2520T>C and c.2589G>A) that did not change the polypeptide sequence and, therefore, are unlikely to be causative for CPA. Indeed, the two SNPs of the diseased piglets are also represented in normal human variants, clearly implying a non-disease association.

Real-time PCR was performed using SYBR green dye on an Applied Biosystems 7500 Real Time System. Samples were run in triplicate and the averaged C_t-values normalized to those of the housekeeping gene 18S. The ratio between the two groups of piglets was calculated with the ΔΔC_t method;⁷ the resulting value of 0.56 indicated that the expression of INPP4A in quadriceps muscle did not differ between affected and normal piglets.

Comments: In summary, SLC5A7 was excluded as a CPA candidate gene based on RH mapping, while the results based on mutation analysis and gene expression excluded INPP4A from causal association. In addition, the mapping results reported highlight additional chromosomal homologies to expand and refine the comparative map between HSA2 and SSC3, which will help to identify the CPA gene.