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Mutations in the TCIRG1 gene, coding for a subunit of the osteoclast proton pump, are responsible for more than 50% of cases of human malignant autosomal recessive osteopetrosis (ARO), a rare inherited bone disease with increased bone density owing to a failure in bone resorption. A wide variety of mutations has been described, including missense, nonsense, small deletions/insertions, splice-site mutations, and large genomic deletions, all leading to a similar severe presentation. So far, to the best of our knowledge, no report of a mild phenotype owing to recessive TCIRG1 mutations is present neither in our series of more than 100 TCIRG1-dependent ARO patients nor in the literature. Here we describe an 8-year-old patient referred to us with a clinical diagnosis of ARO, based on radiological findings; of note, no neurological or hematological defects were present in this girl. Surprisingly, we identified a novel nucleotide change in intron 15 of the TCIRG1 gene at the homozygous state, leading to the production of multiple aberrant transcripts, but also, more importantly, of a limited amount of the normal transcript. Our results show that a low level of normal TCIRG1 protein can dampen the clinical presentation of TCIRG1-dependent ARO. On this basis, a small amount of protein might be sufficient to rescue, at least partially, the severe ARO phenotype, and this is particularly important when gene therapy approaches are considered. In addition, we would also recommend that the TCIRG1 gene be included in the molecular diagnosis of mild forms of human ARO., (© 2014 American Society for Bone and Mineral Research.)

Unlabelled: Here we report 41 novel mutations in the TCIRG1 gene that is responsible for the disease in more than 50% of ARO patients. The characterisation of mutations in this gene might be useful in the process of drug design for osteoporosis treatment., Introduction: Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder due to reduced bone resorption by osteoclasts. In this process, a crucial role is played by the proton pump V-ATPase. Biallelic mutations in the TCIRG1 gene, encoding for the a3 subunit of this pump, are responsible for more than one half of ARO patients., Methods: Patients with a clinical diagnosis of ARO have been collected for 7 years and mutation analysis of the TCIRG1 gene was performed using direct DNA sequencing of PCR-amplified exons according to both a standard protocol and a modified one., Results: We report here 41 novel mutations identified in 67 unpublished patients, all with biallelic mutations. In particular, we describe two novel large genomic deletions and two splice site mutations in the 5' UTR of the TCIRG1 gene, in patients previously classified as mono-allelic., Conclusions: Our data highlights the importance of two large genomic deletions and mutations in the 5' UTR with respect to patient management and, more critically, to prenatal diagnosis. With the present work, we strongly contribute to the molecular dissection of TCIRG1-deficient ARO and identify several protein residues which are fundamental for proton pump function and could thus be the target of future drugs designed to inhibit osteoclast resorptive activity.

Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Biallelic mutations in the TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, are responsible for more than one half of ARO patients. However, a few patients with monoallelic mutations have been described, raising the possibility of a dominant-like TCIRG1-dependent osteopetrosis, of a digenic disease, or of peculiar mutations difficult to detect with standard methods. We describe here a novel genomic deletion in the TCIRG1 gene explaining why, in some patients, mutations in only one allele have previously been found. The analysis of a proband from a consanguineous Turkish family allowed us to define the deletion boundaries encompassing introns 10 and 13 and occurring within AluSx repeat sequences, suggesting Alu-mediated homologous recombination as a mechanism. An identical genomic deletion at the heterozygous level was found in four unrelated Italian families in whom only a single mutated allele has previously been found. TCIRG1 haplotype analysis in these five families suggests a possible common ancestral origin for this large deletion. In summary, we describe the identification of a novel genomic deletion in the TCIRG1 gene that is of clinical relevance, especially in prenatal diagnosis.