Identification and characterization of the molecular lesion causing mucopolysaccharidosis type I in cats

Mucopolysaccharidosis Type I (MPS I) is the lysosomal storage disease cause d by the deficient activity of alpha-L-iduronidase (IDUA). In man, MPS I ca n occur in severe, mild, or intermediate forms known as the Hurler, Scheie, or Hurler/Scheie syndromes, respectively. RIPS I also has been described i n cats, dogs, and mice. This manuscript reports the identification and char acterization of the mutation causing RIPS I in cats. To obtain wild-type fe line IDUA cDNAs, two PCR-based strategies were used. PCR primers were const ructed from a conserved region of the published human and dog sequences and used to amplify a 224-bp IDUA fragment from normal cat genomic DNA. This f ragment was then used to screen a feline uterus cDNA library. PCR also was used to directly amplify IDUA fragments from the same cDNA library. Two ove rlapping feline IDUA cDNAs encoding 466 amino acid residues of the feline I DUA polypeptide (similar to 85% of the mature protein based on comparison t o the human, dog, and mouse sequences) were obtained by these strategies. T o identify the mutation causing NIPS I in cats, DNA sequencing was carried out on the corresponding IDUA region from several affected animals. A 3-bp deletion was found on both IDUA alleles in each of the MPS I animals, predi cting the deletion of a single aspartate residue from the feline IDUA polyp eptide. To confirm the authenticity of this mutation, heteroduplex, SSCP, a nd transient expression studies were carried out. Over 100 animals from the RIPS I colony were screened for the presence of the mutation by heterodupl ex and SSCP analyses-in all cases the presence of the 3-bp deletion was 100 % concordant with the disease phenotype. For transient expression studies, the two partial, overlapping feline cDNAs were combined and joined in-frame to the 5' end of the canine IDUA cDNA. This wild-type, hybrid cDNA express ed IDUA activity up to sixfold over endogenous levels after transfection in to COS-1 cells. A modified full-length IDUA cDNA containing the 3-bp deleti on did not express IDUA activity in a transient expression system, providin g proof that this lesion was the cause of feline MPS I. (C) 1999 Academic P ress.