Lymphocyte mRNA analysis of the ornithine transcarbamylase gene in Italian OTCD male patients and manifesting carriers: Identification of novel mutations

A new simple, non-invasive method using ornithine transcarbamylase (OTC) mRNA isolated from peripheral blood (PBL) or lymphoblastoid cell lines has been performed. This approach based on reverse transcription and nested PCR to obtain a double strand PBL OTC cDNA allowed the identification of genetic lesions in five Italian families affected by OTC deficiency (OTCD). In the PBL OTC mRNA two new mutations, T262K and W265L, have been detected in three unrelated male OTCD patients with mild symptoms. One known mutation, T264A, has been identified in one manifesting carrier. The known mutation E310X, detected on genomic DNA of another manifesting carrier, failed to be detected in her PBL OTC mRNA because of the presence of a STOP codon. All mutations have been confirmed in the patients' and their relatives' genomic DNA. In three patients the mutations have also been confirmed in the mRNA isolated from frozen liver biopsy. The T262K amino acid substitution has been detected in a male's PBL OTC mRNA at homozygous state while a heterozygous pattern has been detected at the genomic DNA level, suggesting that the patient is a somatic mosaic for this mutation. Here we show that PBL OTC mRNA analysis is useful to detect genetic lesions in male and female OTCD patients