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Fifteen years of genetic dissection of human osteopetrosis have greatly extended our understanding of the physiopathology of this disease and, more in general, of the bone tissue. At present, an exact classification is obtained in most cases of the extreme phenotypes: on one hand, autosomal recessive forms with a very severe and early presentation; on the other, adult, benign forms mildly impacting on the patients' life. This allows the identification of the most appropriate therapy. On the contrary, intermediate, atypical forms are more difficult to solve and manage; therefore, these are often addressed by next-generation sequencing technologies or require an adaptation of commonly used protocols.

We recently reported the identification of an incomplete splicing defect in the TCIRG1 gene leading to benign, recessive osteopetrosis in a young girl.[2] At that time, we thought that this kind of phenotype owing to the TCIRG1 gene was extremely rare because no other similar finding was present in either our cohort of more than 300 patients or in the literature, to the best of our knowledge. However, our present data suggest that this is not the case; in fact, we report here the identification of 4 different single nucleotide changes in intron 15 predicted to impact on the splicing process of the neighboring exons. For 2 of 4 novel mutations, the effect was demonstrated at the cDNA, showing that these genetic defects were hypomorphic and allowed for the production of a small amount of wild-type transcript. In addition, bone histology in Pt 1A allowed us to clearly document osteoclast-rich osteopetrosis, in the presence of areas of well-formed, mature trabecular bone. Interestingly, the mutations herein described are located in close proximity to one another and probably identify a hot spot region for mutation within intron 15. Moreover, they are found in the middle of a 368-nucleotide-long intron, quite far from the canonical splice sites, so our standard molecular analysis of the TCIRG1 gene initially failed to identify them. Due to their localization, we were not able to exactly define the pathogenic mechanism; however, a careful sequence analysis of the alternative transcripts revealed the usage of cryptic splicing sites, namely an acceptor in exon 15 and a donor in intron 15. Deep intronic mutations, leading to the formation of cryptic splice sites which compete with the canonical ones during RNA processing, have been recently reported also in different diseases, such as Stargardt disease, Gorlin syndrome, optic atrophy, Marfan syndrome and others.[23-26] In the field of skeletal diseases, a deep intronic mutation in the TMEM165 gene has been reported to cause the congenital disorder of glycosylation type II with bone dysplasia.[27] At present this type of defects is likely underestimated, since the genetic tests are still mainly focused on coding genomic sequences and transcript analysis is rarely performed; however, these data clearly highlight the importance of the noncoding genome and the need to assess the functional impact of remote intronic variants in potential disease-genes.

The patients described in the present study displayed a different level of severity of the disease. Whereas in Pt 3 and Pt 4 the diagnosis was made in early life and their osteopetrosis was treated with HSCT, in Pt 1A and Pt 2 the bone defects became apparent at school age and the disease progression was slow and overall benign, thus representing typical intermediate cases. Moreover, Pt 1B was diagnosed at an asymptomatic early stage because of the previous identification of his elder affected sister, Pt 1A. In this regard, the difference between these two siblings is interesting; whereas the elder showed important secondary neurological deficits, these were completely absent in her younger brother, who seemed to be more mildly affected. This is reminiscent of what is commonly observed in autosomal dominant osteopetrosis, in which phenotypic variations can be observed in the presence of the very same genetic defect, even within the same family. A role for variants in modifier genes has been often suggested. Intriguingly, we noticed that Pt 1A and Pt 1B carried an almost completely different set of single-nucleotide polymorphisms (SNPs) in the CLCN7 gene (only 2 shared SNPs out of 30 genotyped, data not shown). Among these, Pt 1B, but not his sister, bore 2 SNPs within exon 15 (rs12926089 and rs12926669) at the heterozygous state, which have been associated with low bone mineral density in different populations.[28, 29] Similarly, it could be hypothesized that additional variants in other genes influencing bone metabolism are present in one or the other affected sibling and modulated their phenotype. Moreover, in adolescence and adult life, sexual hormones might have further impacted on the disease manifestation.

In conclusion, our results depict an even wider spectrum of mutations and phenotypes in TCIRG1-dependent osteopetrosis and suggest the analysis of this gene is appropriate not only in the molecular work-up of severe patients but also of intermediate cases. Based on these findings, standard protocols for gene amplification and sequencing, focused on exons and exon-intron boundaries, are likely to be revised. In particular, intron 15 should be included in the routine sequencing of the TCIRG1 gene; more in general, intronic changes in genes associated with osteopetrosis, even deeply embedded in introns, should be considered and their effect carefully evaluated.

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GAP II è un sistema per l’archiviazione di dati clinici e chirurgici di pazienti sottoposti ad interventi di chirurgia protesica di anca, ginocchio e spalla.