Clinical evaluation of patients

Patients 1A and 1B (Pt 1A and Pt 1B) were two affected siblings born from Italian consanguineous parents (third-degree consanguinity). Pt 1A, female, was first evaluated at age 6 years because of visual problems, in particular right amaurosis and divergent strabismus. A radiological investigation of the skull revealed increased bone density, and a skeletal survey confirmed generalized osteopetrosis (Fig. 1A). Computed tomography (CT) scan showed bilateral optic and acoustic foramina restriction; analysis of the visual evoked potentials (VEP) confirmed visual impairment on the right eye, whereas brainstem evoked response audiometry (BERA) gave normal results. At that time, her hematological function was normal despite mild hepatosplenomegaly. On these results, a diagnosis of osteopetrosis with low progression was made. The presence of a similar bone phenotype in her younger brother (Pt 1B) at the age of 2 years (Fig. 1B), together with the absence of pathological findings in their parents as well as their consanguinity, suggested an autosomal recessive pattern of inheritance. In the following years, the clinical history of both patients was mainly characterized by recurrent fractures (8 in Pt 1A, 11 in Pt 1B) at different skeletal segments, particularly the lower limbs (Fig. 1C), and dental problems, with delayed tooth eruption, congenitally missing teeth and, in the elder sibling, dental abscesses, requiring intravenous administration of antibiotics and a surgical intervention. The visual acuity progressively worsened in Pt 1A and was completely lost in the right eye side at the age of 9 years; subsequently, a surgery was performed to preserve the vision of the left eye. In addition, she lost hearing in the left ear starting from age 6 years. Most recent laboratory investigations, performed concomitantly with an orthopedic surgery for total hip replacement after removal of a previous prosthesis, did not show alterations of the evaluated parameters (Table 1). Histological analysis on a bone specimen from Pt 1A demonstrated an incomplete osteopetrosis with some areas displaying mostly retained mineralized cartilage and primary spongiosa, whereas others showing mostly normal lamellar bone in well-formed trabecular bone (Fig. 2). Importantly, several osteoclasts were visualized, thus clearly identifying the disease in this patient as an osteoclast-rich osteopetrosis (Fig. 2). At present, both affected siblings do not receive any specific therapy. Overall, their clinical picture can be considered benign, with Pt 1B displaying no sensorial defects and leading an almost completely normal life at age 32 years.

Fig. 1
Fig. 1.

Representative X-rays of Pt 1A, Pt 1B, and Pt 2, showing a diffuse increase in bone density, the typical harlequin-mask pattern of the sclerotic skull base on the AP cranium, the lack of metaphyseal bone remodeling, and the bone deformities resulting from the remodeling defects and the non-perfect healing and remodeling of fractures. (A) Pt 1A at age 6 years; (B) Pt 1B at age 2 years; (C) Pt 1A at age 35 years; (D) Pt 2 at age 22 years. Overall, the radiographs testify for a severe form osteopetrosis..

Table 1.  Most Recent Laboratory Data in Pt 1A andPt 1B
 Pt 1APt 1BRef. range
Age (years) 37 33  
Erythrocytes (× 106/μL) 3.79 5.10 4.2–5.4
Hemoglobin (g/dL) 11.2 14.5 12–16
Hematocrit (%) 34.5 43.9 37–47
Serum calcium (mmol/L) 2.38 2.48 2.10–2.60
Serum phosphorus (mmol/L) 1.22 1.40 0.80–1.50
Serum iron (μg/dL) 98 123 50–170
Serum ferritin (ng/mL) 23 288 10–120
Serum alkaline phosphatase (U/L) 44 65 40–150
Serum bone ALP isoenzyme (μg/L) 6.2 10.9 6–26
Serum osteocalcin (ng/mL) 21 18 6.5–42.3
Serum PTH (pg/mL) 37.6 27.1 14.5–87.1
Serum calcitonin (pg/mL) 3.8 4 0–5.5
Serum 25(OH)D3 (ng/mL) 10.4 23.2 8.6–54.8
Fig. 2
Fig. 2.

Representative histological sections of a bone specimen from Pt 1A at age 35 years. (A) Visualization in polarized light and hematoxilin-eosin staining. Scale bar = 200 μm. The mature cortical bone shows lamellae in polarized light; the defects within the bone are islands of retained mineralized cartilage. (B) Higher magnification of hematoxilin-eosin staining showing several multinucleated osteoclasts. Scale bar = 100 μm. (C) TRAP fluorescent antibody labeling (red) and phase contrast with the fluorescence image overlaid; nuclei are stained with Hoechst (blue). The inset shows a single osteoclast at twice the magnification. Scale bar = 100 μm; inset = 10 μm. (D) TCIRG1 fluorescent antibody labeling (green) and phase contrast with the fluorescence overlaid (right). Scale bar = 100 μm; inset = 10 μm.

Patient 2 (Pt 2) was the third child of unrelated, healthy Italian parents. The symptoms of the disease appeared at age 4 years, with a generalized increase in bone density (representative, most recent radiological findings in Fig. 1D). He reported 7 nontraumatic fractures and suffered from recurrent osteomyelitis of the jaw. He underwent 3 orthopedic surgeries and several maxilla-facial interventions. No hematological nor sensorial deficits arose, and the patient is alive and reasonably well at age 24 years.

Patient 3 (Pt 3) has been already described (Pt 6 in Mazzolari and colleagues[17]). Briefly, the patient presented with a classical ARO phenotype, with bone defects and secondary hematological and neurological deficits at age 6.5 months. She received hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD) at age 20.5 months, achieved full engraftment, and was alive and well 7.5 years after transplantation.

Patient 4 (Pt 4) was born from healthy unrelated parents from Southeastern Asia. He presented with bone defects, blindness, and hepatosplenomegaly in neonatal life. He received HSCT but eventually died 4 years after HSCT without engraftment. No further information is available.

Genetic findings

Based on the suspected recessive pattern of inheritance of the disease in Pt 1A and Pt 1B, molecular analysis in this family initially focused on the genes known to cause recessive osteopetrosis (TCIRG1, CLCN7, PLEKHM1, RANKL, RANK, and SNX10); the OSTM1 gene was not included in the analysis because the two affected siblings did not display the primary neurological defects that are a hallmark of this ARO form. However, no molecular defect could be identified; therefore, we performed exome sequencing on both patients and their parents. This approach achieved an 80× mean coverage over the 62 Mb of exomic sequence, with more than 94% of targeted regions covered. The overall transition to transversion rate (Ti/Tv) was 2.4, which is in line with what was expected for exome sequencing. The analysis identified a total of 191,872 variants that were filtered with dbSNP138 and 1000 Genome Project and according to the pattern of inheritance of the disease and to the parental consanguinity (Table 2).

Table 2. Number of Variants/Genes Identified in Pt 1A and Pt 1B Through Exome Sequencing
Total variants 191,872
Shared variants after variant-quality filtering 157,065
Variant with MAF <0.05 19,170
Nonsynonymous/indel/splice sites/UTR variants 1319/98/506/5149
Homozygous variants 32
Novel variants (dbSNP138/1000GP queried) 2
Genes with plausible disease association 1 (LRP5)

Among the homozygous variants, we found a single nucleotide change in exon 22 of the LRP5 gene (Supplementary Fig. 1A) (NG_015835.1:g.138882C>T; NM_002335:c.4574C>T) predicted to cause an amino acid substitution (p.Ala1525Val). Thus far, the so-called “activating mutations” linked with an increased bone density are clustered in the first beta-propeller, at the extracellular N-terminus of the protein, whereas mutations causing low bone density appear to be clustered in the second beta-propeller.[ 18] Interestingly, the variant c.4574C>T lies in a completely different region of the protein, that is the cytoplasmic tail (Supplementary Fig. 1B), known to interact with axin and FRAT, which are key components in the Wnt/β-catenin signaling pathway.[ 19, 20]

So we confirmed this variant by Sanger sequencing and tested its activity in a classical luciferase reporter assay; however, the activation of the Wnt signaling was comparable in the presence of either the wild-type or the mutant LRP5 construct (Supplementary Fig. 1C). In addition, in this experiment, no difference between the wild-type and mutant LRP5 was observed after cotransfection with sclerostin (Supplementary Fig. 1C), whereas the “activating mutations” in LRP5 are known to result in increased Wnt signaling activity owing to reduced inhibition by sclerostin or dickkopf 1. So, these results did not support the hypothesis of a causative role for the variant in the pathogenesis of the disease. Accordingly, at the time when this article was in preparation, an update of SNP databases included this change in the LRP5 gene at the homozygous state.

Therefore, we returned to the list of variants identified by exome sequencing. No additional noteworthy variants in coding regions were present, so we focused our attention on variants in 3′ untranslated regions (3′UTR) because they are known to influence gene expression.[21] A single nucleotide change was annotated in the 3′UTR of the choline kinase alpha (CHKA) gene (NM_001277; *327T>C), at the homozygous state in patients 1A and 1B and at the heterozygous state in their parents. Interestingly, CHKA maps to chromosome 11q13.2 on the reverse strand and immediately downstream to the TCIRG1 gene. Because of the close proximity of the two genes, we asked whether the *327T>C variant might affect the expression of the TCIRG1 gene. Investigation at the cDNA level through semiquantitative RT-PCR did not show reduced expression; however, at least two unexpected bands in the two affected siblings by RT-PCR spanning exons 14 to 17 were observed (Fig. 3A), suggesting a splicing aberration. Therefore, we sequenced the corresponding genomic region, including also the whole of the three introns, and identified a single nucleotide change in the middle of intron 15 (g.10466G>A, c.1884+146G>A), which was not reported in dbSNP and was present at the homozygous state in Pt 1A and Pt 1B and at the heterozygous state in their parents but absent in 100 control chromosomes from a matched population (Fig. 3B and data not shown). Despite this change being far from both the donor of exon 15 and the acceptor of exon 16, in silico analysis suggested a potential detrimental effect on the splicing process, by altering the strength of alternative splice sites compared with the wild-type sequence. As a confirmation of this prediction, when we cloned the cDNA PCR product spanning exons 14 to 17 and sequenced 73 independent clones, we found that few clones displayed the wild-type sequence (4/73 clones), whereas at least 8 diverse aberrant transcripts were produced, the most abundant being clones retaining 144 nucleotides of intron 15 (22/73 clones) and clones in which the first 183 nucleotides of exon 15 were skipped and 144 nucleotides of intron 15 retained (30/73 clones) (Fig. 3C). These data clearly supported the hypothesis of a pathogenetic role of the variant.

Fig. 3
Fig. 3.

(A) Semi-quantitative RT-PCR spanning exons 14 to 17 (upper panel), showing the presence of at least two abnormal bands in the patients (the normal band seen in Pt 2 is from the other mutated allele), or exons 7 to 10 (middle panel), showing only the presence of the expected band in all the samples. (B) Schematic representation of the relevant region of the TCIRG1 gene, highlighting the position of the mutations identified in intron 15; the nucleotide change found in each patient is in bold in the lower line. (C) Structure of the most frequent aberrant transcripts found in Pt 1A, Pt 1B and Pt 2: i) clones in which only the last 31 nucleotides of exon 15 are retained, followed by 143 nucleotides of intron 15, and ii) clones retaining 143 nucleotides of intron 15. The nucleotide change found in each patient is in bold in the lower line, as in panel B.

Prompted by these findings, we sequenced TCIRG1 intron 15 in 26 osteopetrotic patients not yet assigned to any subgroup because of the absence of mutations in known genes, as well as in 7 ARO patients in which we had previously identified only a single mutated TCIRG1 allele[17, 22] (unpublished data). Although no mutation was found in the 26 unclassified patients, in 3 of the 7 patients harboring a single mutated TCIRG1 allele, we found 3 additional, heterozygous single nucleotide changes in intron 15, close to that carried by Pt 1A and Pt 1B, namely g.10462T>A (c.1884+142T>A) in Pt 2 (mutation confirmed at the heterozygous state in Pt 2's healthy mother and in one unaffected brother; data not shown), g.10452T>C (c.1884+132T>C) in Pt 3, and g.10469C>T (c.1884+149C>T) in Pt 4 (Fig. 3B and Table 3. In silico analysis was performed also on these variants, obtaining a similar prediction of potential disruptive effects on the splicing process. A cell sample suitable for expression studies was available only for Pt 2, and we could confirm also in this case by RT-PCR followed by cloning and sequencing, the presence of a residual amount of wild-type transcript (5/75 clones, 6.7%) and the same pattern of aberrant transcripts previously found in Pt 1A and Pt 1B, namely 12/75 clones retaining 143 nucleotides of intron 15, 20/75 clones skipping the first 183 nucleotides of exon 15 and retaining 143 nucleotides of intron 15, 20/75 clones skipping the first 183 nucleotides of exon 15 and retaining 141 nucleotides of intron 15, plus other less frequent species. Sequence analysis of individual clones showed that loss of large part of exon 15 was due to the usage of an internal, cryptic acceptor site, while retention of 143 nucleotides of intron 15 was due to the usage of an intronic, cryptic donor site (Supplementary Fig. 2).

Table 3.  Molecular Findings
PatientGenomic changeaLocationcDNA changebEffect
  • a

    Accession number genomic sequence of the TCIRG1 gene: AF033033.

  • b

    Accession number of the TCIRG1variant1 cDNA: NM_006019; the numbering used starts with nucleotide +1 for the A of the ATG-translation initiation codon.

1A and 1B g.10466G>A Intron 15 c.1887+146G>A Putative aberrant splicing
g.10466G>A Intron 15 c.1887+146G>A Putative aberrant splicing
2 g.9909G>A Exon 14 c.1559G>A p.Trp520X
g.10462T>A Intron 15 c.1887+142T>A Putative aberrant splicing
3 g.11622T>C Exon 19 c.2345T>C p.Met782Thr
g.10452T>C Intron 15 c.1887+132T>C Putative aberrant splicing
4 g.8600T>C Intron 11 c.1305+2T>C Putative aberrant splicing
g.10469C>T Intron 15 c.1887+149C>T Putative aberrant splicing

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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.