Genetic analysis in nine unrelated Italian patients affected by OTC deficiency: detection of novel mutations in the OTC gene

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Abstract

Ornithine transcarbamylase deficiency (OTCD) is an X-linked urea cycle disorder due to a defect of the mithocondrial enzyme ornithine transcarbamylase (OTC). Genetic analysis in nine unrelated Italian patients affected by OTCD (one male patient and eight female manifesting carriers) led to the detection of three novel mutations and six previously reported mutations in the OTC gene. The analysis was performed by direct sequencing of OTC cDNA, OTC exons, and intron–exon boundaries and enzymatic restriction analysis on the patients' genomic DNA and total RNA isolated from peripheral blood lymphocytes. In the male patient the new mutation S132P due to the nucleotide change c.394T>C was identified. In a manifesting carrier the nucleotide change c.292G>A that leads to the novel amino acid substitution E98K was identified; this mutation is close to the OTC protein's carbamyl phospate binding site. In another manifesting carrier the OTC cDNA analysis revealed the normally spliced transcript and an aberrant transcript with an insertion of two nucleotides (c.77–78insAG). In the patient's genomic DNA we identified a new transvertion IVS1−3C>G at the heterozygous state; this nucleotide change generates a new splice acceptor site in intron 1 that induces an RNA splicing defect. This insertion causes a frame shift in OTC cDNA ORF and leads to a premature stop codon. The previously described mutations N161S, R141Q, T178M, R92X, A208T, M268T were identified in the other six manifesting carriers.

Introduction

Ornithine transcarbamylase deficiency (OTCD; MIM# 311250) is the most common urea cycle disorder, caused by a defect of the mithocondrial enzyme ornithine transcarbamylase (OTC, EC 2.1.3.3), which is expressed specifically in the liver and small intestine [1]; it is inherited as an X-linked trait.

The OTC gene is subject to heterogeneous and frequent new mutations, which may lead to partial or complete enzymatic deficiency. Clinical presentation is highly variable, usually more severe in newborn male patients. In hemizygous male patients the onset of the disease can also occur later in infancy, during childhood or even in adult life with varying degrees of severity, which is principally correlated to different mutations and enzyme activity. In heterozygous female manifesting carriers the clinical phenotype depends on X-inactivation [2] and it ranges from asymptomatic to severe hyperammonemia.

The structural gene encoding for the enzyme OTC has been mapped to the short arm of the X chromosome at band Xp21.1 [3]. It spans a region of 73 kb and contains 10 exons and 9 introns of highly variable size [4]. The OTC mRNA contains 1062 nt and it encodes a 40 kDa protein precursor of 354 amino acids which includes the amino terminus leader peptide of 32 amino acids [5].

Up to now about 230 mutations in the OTC gene have been reported (http://63.75.201.100/otc/) [6], most of which are “private” mutations [7]. Large deletions of one exon or more are seen in approximately 7% of patients, small deletions or insertions are seen in about 9%, and the remaining mutations are single base substitutions; approximately 15% of mutations affect RNA splicing sites [8].

In this study genetic analysis by direct sequencing of OTC cDNA, genomic DNA, and enzymatic restriction analysis on the patients' genomic DNA and total RNA isolated from peripheral blood lymphocytes led to the identification of three new mutations and six previously reported mutations in the OTC gene of nine unrelated Italian patients and their families. Interestingly, eight of the nine patients were females. These patients came from different hospitals, they were not selected and they represented a casual sampling.

Section snippets

Patients

One OTCD male patient (1.1) and eight OTCD manifesting carriers (2.1, 3.1, 4.1, 5.1, 6.1, 7.1, 8.1, 9.1), segregating in nine unrelated families, were studied. The male patient with the mild form and onset at three years showed classical OTCD clinical symptoms (vomiting, confusion, lethargy) and characteristic laboratory findings (high ammonemia, high glutaminemia, low citrullinemia, and high urinary orotic acid excretion). The manifesting carriers presented clinical heterogeneity varying from

Results and discussion

The OTC gene is subject to heterogeneous and frequent novel genetic lesions, most of which are “private” mutations, that may lead to partial or complete OTC enzymatic deficiency. The high new mutation rate and the wide spectrum of mutations mean that direct mutation analysis is essential for the identification of the causative genetic lesion in affected families.

Genetic analysis in one OTCD male patient and eight female manifesting carriers from nine unrelated Italian families led to the

Acknowledgements

This work was partially supported by grants from the Cassa di Risparmio di Firenze, Association AMMEC and Azienda Ospedaliera Meyer.

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    The authors S. Bisanzi and A. Morrone contributed to this work equally.

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