Buried in the Middle but Guilty: Intronic Mutations in the TCIRG1 Gene Cause Human Autosomal Recessive Osteopetrosis

Materials and Methods


Clinical data and specimens, including blood and DNA samples, were collected from patients and their relatives after informed consent. This research complies with the standards established by the Independent Ethical Committee of the Humanitas Clinical and Research Centre.

Exome analysis

Exome capture was performed on 1.5 μg of high-quality genomic DNA from patients 1A and 1B and their parents, and the TruSeq Exome Enrichment Kit (Illumina Inc., San Diego, CA, USA) was used. The enriched library was validated by the Agilent DNA 1000 Kit (Agilent Technologies Inc., Santa Clara, CA, USA) and loaded on the cBot Station (Illumina Inc.) to create clonal clusters on the flow cell. Sequencing was performed on the Hiseq2000 Instrument. Reads extracted with the Illumina tools were aligned to the reference genome hg19 by using BWA-MEM[4] and stored in compressed binary files (BAM). Single nucleotide variations, insertions, and deletions were called using the Genome Analysis Toolkit (GATK).[5] All the analyses were performed using the Orione platform.[6] The variants identified were merged in a VariantCallingFormat (VCF) file, annotated by KGGSeq,[7] prioritized, and filtered according to a standard workflow for exome sequencing.

Molecular studies

The molecular analysis of genes known to be responsible for the different types of the disease (TCIRG1, CLCN7, PLEKHM1, RANKL, RANK, and SNX10) was performed by amplification and direct sequencing of exons and intron-exon boundaries as previously described.[ 8-13] The TCIRG1 intron 15 was amplified using the forward primer 10271F 5′-TGTTCCTCTTCTCCCACAGC-3′ located in exon 15 and the reverse primer 5R 5′-CATCGGAGCTCCAGCCATT-3′ in exon 17; sequencing was performed using the 10271F primer. After mRNA isolation from peripheral blood mononuclear cells lysed in Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcription using the High Capacity cDNA Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturers’ instructions, the effect of the intronic variants on TCIRG1 transcript processing was investigated with the forward primer 9912F 5′-GCCTGGCTGCCAACCACTTG-3′ in exon 14 and the reverse primer 5R in exon 17. For patient (Pt) 1A and Pt 1B, the PCR product was directly cloned in the TOPO TA Cloning plasmid vector (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions, and individual clones were sequenced using the 9912F forward primer. For Pt 2, a PCR product was obtained from the cDNA with the forward primer 5F 5′-CTGGCCCAGCACACGATGCT-3′ in exon 13 and the reverse primer 5R in exon 17; this product was cloned and individual clones were sequenced using primer 5R. For the confirmation of the variant in the LRP5 gene, the relevant genomic region was amplified using primers forward 5′-TGGGAGGAAGGAAGGAATGC-3′ and reverse 5′-TCAGTGGCATGGGGATTAGG-3′, whereas primers forward 5′-GGGAGTGAGCACCGTCTATA-3′ and reverse 5′-CTCCTAGGACTACGCCCAAG-3′ were used for confirmation of the variant in the CHKA gene; in both cases, sequencing was performed using the forward primer.

For all the PCR reactions above, the thermocycling conditions were: initial denaturation step at 94°C for 3 minutes, followed by 34 cycles of denaturation at 94°C for 30 seconds, annealing at 62°C (55°C for the LRP5 variant) for 30 seconds, and amplification at 72°C for 30 seconds.

The effect of the mutations identified in the intron 15 of the TCIRG1 gene was predicted using the software Human Splicing Finder, Version 2.4.1 (www.umd.be/HSF).[14] The mutation nomenclature conforms to HGVS (www.hgvs.org/mutnomen).[15]

Histological analysis

A bone biopsy specimen of Pt 1A was fixed in 4% paraformaldehyde (PFA), decalcified for 4 hours at 20 °C in 5% HCl (pH ∼1), then rinsed in water, paraffin-embedded, and sectioned at 6 μm. Sections were deparaffinized for hematoxylin-eosin staining and for fluorescent antibody labeling as described.[16] For TCIRG1 labeling, a mouse monoclonal (clone 6H3) antibody was used (Sigma Aldrich Co., St. Louis, MO, USA) at 1:100 dilution. For TRAP, a rabbit polyclonal antibody, C-terminal (Abcam PLC, Cambridge, UK) was used at 1:100 dilution. Briefly, sections were blocked in PBS/2% BSA for 2 hours, reacted overnight with antibodies at indicated concentrations in PBS with 0.01% Tween 20, then labeled for fluorescent analysis using Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody (green) or Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody (red) antibodies, at 1:1000, both from Life Technologies, for 1 hour. For nuclear labeling, Hoechst 33342 blue (Invitrogen) was used. A Nikon TE2000 inverted microscope was used for imaging, via 14-bit 2048 × 2048 pixel monochrome CCD and RGB filters to reconstruct color (Spot, Sterling Heights, MI, USA). Green fluorescence (Cy-2) used excitation at 450 to 490 nm, a 510-nm dichroic mirror, and a 500- to 570-nm emission filter. Red fluorescence (Cy3) used excitation at 530 to 560 nm, a 575-nm dichroic mirror, and a 580- to 650-nm emission filter.