Molecular defect in siblings with prolidase deficiency and absence or presence of clinical symptoms. A 0.8-kb deletion with breakpoints at the short, direct repeat in the PEPD gene and synthesis of abnormal messenger RNA and inactive polypeptide. Prolidase deficiency is an autosomal recessive disorder with highly variable symptoms, including mental retardation, skin lesions, and abnormalities of collagenous tissues. In Japanese female siblings with polypeptide negative prolidase deficiency, and with different degrees of severity of skin lesions, we noted an abnormal mRNA with skipping of 192 bp sequence corresponding to exon 14 in lymphoblastoid cells taken from these patients. Transfection and expression analyses using the mutant prolidase cDNA revealed that a mutant protein translated from the abnormal mRNA had an Mr of 49,000 and was enzymatically inactive. A 774-bp deletion, including exon 14 was noted in the prolidase gene. The deletion had termini within short, direct repeats ranging in size of 7 bp (CCACCCT). The "slipped mispairing" mechanism may predominate in the generation of the deletion at this locus. This mutation caused a 192-bp in-frame deletion of prolidase mRNA and was inherited from the consanguineous parents. The same mutation caused a different degree of clinical phenotype of prolidase deficiency in this family, therefore factor(s) not related to the PEPD gene product also contribute to development of the clinical symptoms. Identification of mutations in the PEPD gene from subjects with prolidase deficiency provides further insight into the physiological role and structure-function relationship of this biologically important enzyme. 