title: Buried in the Middle but Guilty: Intronic Mutations in the TCIRG1 Gene Cause Human Autosomal Recessive Osteopetrosis.
abstract: Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exome sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes.
authors.affiliation: UOS/IRGB, Milan Unit, National Research Council (CNR), Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Palagano
authors.affiliation: Veteran's Affairs Medical Center and Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Harry C
authors.initials: HC
authors.lastname: Blair
authors.affiliation: UOS/IRGB, Milan Unit, National Research Council (CNR), Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pangrazio
authors.affiliation: Veteran's Affairs Medical Center and Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Tourkova
authors.affiliation: UOS/IRGB, Milan Unit, National Research Council (CNR), Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Dario
authors.initials: D
authors.lastname: Strina
authors.affiliation: CRS4, Science and Technology Park Polaris, Piscina Manna, Pula, Italy.|Institute of Genetic and Biomedical Research (IRGB), National Research Council (CNR), Monserrato, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Angius
authors.affiliation: CRS4, Science and Technology Park Polaris, Piscina Manna, Pula, Italy.
authors.forename: Gianmauro
authors.initials: G
authors.lastname: Cuccuru
authors.affiliation: CRS4, Science and Technology Park Polaris, Piscina Manna, Pula, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Oppo
authors.affiliation: CRS4, Science and Technology Park Polaris, Piscina Manna, Pula, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Uva
authors.affiliation: Department of Medical Genetics, University of Antwerp, Antwerp, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Van Hul
authors.affiliation: Department of Medical Genetics, University of Antwerp, Antwerp, Belgium.
authors.forename: Eveline
authors.initials: E
authors.lastname: Boudin
authors.affiliation: Department of Pediatrics, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Superti-Furga
authors.affiliation: Institute for Maternal and Child Health-IRCCS "Burlo Garofolo", Trieste, Italy.
authors.forename: Flavio
authors.initials: F
authors.lastname: Faletra
authors.affiliation: Clinica Pediatrica, Azienda Ospedaliero-Universitaria "S Maria della Misericordia", Udine, Italy.
authors.forename: Agostino
authors.initials: A
authors.lastname: Nocerino
authors.affiliation: Hip and Prosthetic Replacement Unit, Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Matteo C
authors.initials: MC
authors.lastname: Ferrari
authors.affiliation: Hip and Prosthetic Replacement Unit, Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Grappiolo
authors.affiliation: Clinical Investigation Laboratory, Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Monari
authors.affiliation: Clinical Investigation Laboratory, Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Montanelli
authors.affiliation: UOS/IRGB, Milan Unit, National Research Council (CNR), Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Vezzoni
authors.affiliation: UOS/IRGB, Milan Unit, National Research Council (CNR), Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Villa
authors.affiliation: UOS/IRGB, Milan Unit, National Research Council (CNR), Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Sobacchi
mesh_terms: D000328:Adult; D005260:Female; D030342:Genetic Diseases, Inborn / Q000000981:diagnostic imaging / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007438:Introns; D008297:Male; D010022:Osteopetrosis / Q000000981:diagnostic imaging / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D022821:RNA Splice Sites; D011859:Radiography; D025262:Vacuolar Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D022821:RNA Splice Sites; C472421:TCIRG1 protein, human; D025262:Vacuolar Proton-Translocating ATPases
keywords: AUTOSOMAL RECESSIVE OSTEOPETROSIS; EXOME; HYPOMORPHIC MUTATION; SPLICING DEFECT; TCIRG1