Genetics of osteopetrosis: Molecular insights and clinical implications

Autosomal dominant osteopetrosis (OPTA)

Autosomal dominant osteopetrosis is characterized by generalized osteoporosis with a wide intrafamilial and interfamilial clinical variability.^[18,19] A distinctive “bone-in-bone” appearance is a hallmark of OPTA.^[19] It is a late-onset disease with mild clinical features compared to autosomal recessive osteopetrosis (OPTB). Moreover, the disease progression is slow in OPTA. Therefore, OPTA shows a better overall prognosis.^[19] The OPTA has been shown to result from missense variants in the low-density lipoprotein receptor-related protein 5 (LRP5, MIM 603506, and OPTA1) encoding gene,^[20] chloride channel 7 (CLCN7, MIM 602727, and OPTA2) encoding gene,^[21] and pleckstrin homology domain-containing protein, family M, member 1 (PLEKHM1, MIM 611467, and OPTA3) encoding gene.^[22]

• OPTA1 (MIM 607634) is characterized by diffuse, generalized osteosclerosis.^[23] Pronounced sclerosis is observed in the calvaria, and a variable degree of sclerosis is present in the spine region. Sclerosis progresses with age. An increased fracture rate is not observed in OPTA1.^[24] OPTA1 results from activating variants in LPR5, leading to increased osteoblastic bone formation.^[23]

• OPTA2 (MIM 166600), also known as AlbersSchönberg disease, is a late-onset disease.^[21] OPTA2 is characterized by diffuse symmetrical osteosclerosis with increased long-bone fracture rates.^[25] Pronounced skull base sclerosis and hip osteoarthritis are also features of OPTA2. Narrowed nerve canals in the skull due to bone overgrowth compress cranial nerves, often resulting in vision loss, hearing impairment, and facial paralysis. Heterozygous variants in the CLCN7 are the main cause of OPTA2.^[26]

• OPTA3 (OMIM 618107) is characterized by a generalized increase in the density of long bones and the narrowing of the bone marrow cavity due to bone overgrowth, crowding out the marrow space. This causes severe anemia, increased infections, and bleeding disorders. Recurrent fractures with minor trauma are also observed.^[27] The skull in OPTA3 patients is osteosclerotic, characterized by localized osteosclerosis and thickening of the calvarium.^[22] Hepatosplenomegaly and missing teeth are also observed in OPTA3. Heterozygous variants in the PLEKHM1 gene cause the OPTA3 phenotype.^[22,27]

OPTB

OPTB is a more severe form of osteopetrosis and has an early onset. It is a clinically and genetically heterogeneous disorder and is fatal in early childhood if left untreated.^[28] The OPTB have been shown to result from homozygous deletion, splicing, missense, non-sense, and frameshift variants in the T-cell immune regulator 1 (TCIRG1) (TCIRG1, MIM 604592, and OPTB1) encoding gene,^[1] tumor necrosis factor ligand superfamily, member 11 (TNFSF11, MIM 602642, and OPTB2) encoding gene,^[29] carbonic anhydrase II (CA2, MIM 611492, and OPTB3) encoding gene,^[30] chloride channel 7 (CLCN7, MIM 602727, and OPTB4) encoding gene,^[31] osteopetrosis-associated transmembrane protein 1 (OSTM1, MIM 607649, and OPTB5) encoding gene,^[32] pleckstrin homology domain-containing protein, family M, member 1 (PLEKHM1, MIM 611467, OPTB6) encoding gene,^[33] tumor necrosis factor receptor superfamily, member 11A (TNFRSF11A, MIM 603499, and OPTB7) encoding gene,^[34] sorting nexin 10 (SNX10, MIM 614780, and OPTB8) encoding gene,^[35] and solute carrier family 4 (anion exchanger), member 2 (SLC4A2, MIM 109280, and OPTB9).^[36]

• OPTB1 (MIM 259700) is characterized by osteomyelitis, uniformly dense skeleton, fractures, the sandwich appearance of vertebral bodies, coxa vara, facial paralysis, blindness, deafness, and hepatosplenomegaly.^[1,37,38] Biallelic variants in the TCIRG1 gene are known to cause OPTB1.^[1]

• The clinical features in OPTB2 (MIM 259710) include osteosclerosis, osteomyelitis, genu valgum, and hyperostosis of the skull and limbs.^[39] Dental anomalies, including tooth crown malformation, dental caries, and retention of deciduous teeth, are also reported.^[40] Early blindness due to optic atrophy and hepatosplenomegaly is also common features of OPTB2.^[41] Homozygous variants in the TNFSF11 gene cause the OPTB2 phenotype.^[29]

• OPTB3 (MIM 259730) is characterized by postnatal growth retardation, short stature, and multiple fractures.^[42] Moreover, OPTB3 patients show hearing loss, developmental delay, and intellectual disability.^[43,44] Malocclusion, persistent primary teeth, and dental caries are also observed.^[45] Mixed proximal and distal renal tubular acidosis is a hallmark of OPTB3.^[46] Homozygous or compound heterozygous variants in the CA2 gene are the underlying cause of OPTB3.^[47,48]

• OPTB4 (MIM 611490) is a severe form of osteopetrosis with generalized increased bone density, increased trabecular size, and sclerosis of the base of the skull and vertebral endplates.^[49,50] Growth retardation, early onset visual impairment, and hepatosplenomegaly are also observed.^[31] OPTB4 is caused by homozygous or compound heterozygous variants in the CLCN7 gene.^[31,51]

• The most severe form of OPTB is OPTB5 (MIM 259720). The clinical features observed in OPTB5 include growth failure, short stature, generalized increase in bone density, loss of cortico-medullary differentiation, hypocellular bone marrow, reduced bone marrow space, abnormal thickening of the trabeculae, reduced number of osteoclasts, microcephaly, sclerosis of vertebrae, metaphyseal flaring, multiple fractures, hepatosplenomegaly, rib sclerosis, cerebral atrophy, atrophy of corpus callosum and brainstem, and motor nerve conduction abnormalities.^[52-55] OPTB5 is caused by homozygous variants in the OSTM1 gene.^[56-58]

• Clinically, OPTB6 (MIM 611497) is characterized by band-like sclerosis of vertebral endplates (“rugger-jersey” spine), cortical sclerosis of pelvic bones, non-homogeneous sclerosis of metadiaphyses (distal femora, tibiae, and fibulae, and proximal fibulae and tibiae), and “Erlenmeyer flask” deformity of distal femora and proximal tibiae.^[33] Only one published report is available on OPTB6, and homozygous variants in the PLEKHM1 gene lead to the OPTB6 phenotype.^[33]

• Skeletal abnormalities in OPTB7 (MIM 612301) are increased bone density, increased head circumference, thickened bone of vertebrae, multiple fractures, and increased bony and cartilaginous trabeculae. Non-skeletal features include nystagmus, vision loss, atrophy of the optic and olfactory nerves, and hepatosplenomegaly. Psychomotor retardation, motor delay, enlarged lateral ventricles, and hypotonia are also observed.^[34] OPTB7 is caused by homozygous or compound heterozygous variants in the TNFRSF11A gene.^[59]

• The clinical features of OPTB8 (MIM 615085) are macrocephaly, open fontanel, narrow optic and auditory canals, facial nerve palsy, unilateral and/or bilateral vision loss, and hepatosplenomegaly.^[60,61] Variants in the sorting nexin 10 (SNX10) gene are the underlying cause of OPTB8.^[35,62]

• OPTB9 (MIM 620366) is characterized by spontaneous fractures and sclerosis of the skull base, calvarium, vertebral bodies, proximal femur, and ribs.^[36] Extraskeletal features include chronic progressive renal failure, bilateral papilledema, and anemia. Deficiency of solute carrier family 4 member 2 (SLC4A2) is the cause of the OPTB9 phenotype.^[36]

Bone resorption and osteoclast function

The primary theme that interlinks many of these genes (such as CLCN7, TCIRG1, OSTM1, PLEKHM1, SNX10, and CA2) is their involvement in osteoclast function and bone resorption.^[63] For instance, pathogenic variants in PLEKHM1 and SNX10 impair vesicular trafficking within the osteoclasts, a process essential for their proper function.^[64] SNX10 plays a critical role in regulating osteoclast precursor fusion and determining osteoclast size.^[63] Homozygous mutant mice for Snx10 exhibit dysregulated and continuous cell fusion, resulting in the formation of abnormally large, non-functional osteoclasts.^[63] Similarly, mice mutant for the Ostm1 gene disrupts normal protein function during osteoclast maturation, leading to an excessive population of oversized osteoclasts. These findings suggest that Ostm1 can act as a negative regulator of pre-osteoclast fusion.^[65] Osteoclasts are highly specialized cells responsible for bone resorption during the continuous process of skeletal remodeling. This process requires a highly acidic environment in the resorption lacuna. Genes such as CA2, SLC4A2, CLCN7, and TCIRG1 are encoding enzymes and channel proteins that are involved in maintaining the acid-base balance. They maintain the pH necessary for osteoclasts to perform the function of bone resorption. To create an acidic environment, protons (H⁺) are actively transported across the osteoclast membrane by the osteoclast-specific vacuolar-type H⁺-ATPase (V-ATPase) “proton pump.”^[66] The a3 subunit of V-ATPase is encoded by the TCIRG1 gene. In addition, CLCN7 plays a crucial role in facilitating efficient proton pumping by V-ATPase.^[31] CLCN7 encodes chloride voltage-gated channel 7, which functions as a Cl⁻/H⁺ antiporter. This channel works in conjunction with V-ATPase to promote the acidification of the extracellular resorption lacuna, thereby regulating both bone resorption and the processes of bone calcification and degradation.^[46] Moreover, osteoclasts with CA2 deficiency cannot acidify their “sealing zone” due to failure in catalyzing the formation of HCO3⁻ and H+ ions.^[67] Furthermore, SLC4A2 (encoding anion exchanger 2) is involved in exchanging Cl⁻ with HCO3⁻.^[36,68] Variants in SLC4A2 decrease the anion exchange activity of SLC4A2 transporters, thus impairing osteoclastogenesis.^[36,68]

TNFSF11-TNFRSF11A pathway and osteoclast differentiation

The interaction between TNFSF11 and its receptor (TNFRSF11A) is essential for osteoclast differentiation and the process of bone resorption.^[6] Dysregulation of this pathway can lead to osteopetrosis. For instance, deficiency of Tnfsf11 in mice leads to the absence of osteoclasts and osteoblasts.^[69] TNFSF11 is a ligand that is primarily expressed on osteoblasts and stromal cells. It is also expressed in T-cells and dendritic cells. TNFRSF11A is expressed on osteoclasts and triggers a cascade of signaling events on binding with TNFSF11, leading to osteoclast differentiation [Figure 2]. The binding of TNFSF11-TNFRSF11A promotes the differentiation and maturation of osteoclasts, extends their survival, and activates bone resorption through various signaling pathways. ^[46] The binding of TNFSF11 causes conformational changes in the intracellular domain of the TNFRSF11A. This leads to the recruitment of various adaptor proteins, including members of the tumor necrosis factor receptor-receptor-associated factors (TRAFs) family, such as TRAF2 and TRAF6.^[70] This recruitment initiates the activation of several intracellular signaling cascades, including the NF-kB, Mitogen-Activated Protein Kinase (MAPK), and PI3K/Akt signaling cascades.^[71] Activation of the NF-kB cascade involves the phosphorylation and subsequent degradation of the NF-kB inhibitor, resulting in the release and nuclear translocation of NF-kB dimers. These dimers bind to the promoters of target genes, such as those encoding nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), and c-Fos.^[72] All of which are critical regulators of osteoclast differentiation.^[72] NFATc1, in particular, functions as a master transcription factor in osteoclastogenesis. On activation, it translocates to the nucleus. It induces the expression of key osteoclast-specific genes, including TRAP, cathepsin K, and matrix metalloproteinase 9, which are essential for osteoclast differentiation and bone resorptive activity.^[73] Interaction of TNFSF11/TNFRSF11A also activates extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 pathways of the MAPK signaling cascade. ERK, JNK, and p38 pathways upregulate transcription factors and proteins needed for osteoclast formation and fine-tuning of osteoclast differentiation and function, thus promoting osteoclast survival and enhancing bone resorption activity.^[74] Moreover, activation of TNFRSF11A also stimulates the PI3K/Akt cascade, which, in turn, upregulates the expression of anti-apoptotic and survival genes, thereby promoting and enhancing osteoclast survival and function [Figure 3].^[75]

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Figure 2:

Osteoclastogenesis is initiated by tumor necrosis factor superfamily member 11 (TNFSF11)/RANKL signaling from osteoblasts to osteoclast precursors. The schematic illustrates the key cellular interaction that triggers osteoclast formation. Osteoblasts secrete the cytokine TNFSF11 (also known as RANKL). This ligand binds to its specific receptor, TNFRSF11A (also known as RANK), on the surface of osteoclast precursors. This TNFSF11/TNFRSF11A interaction is the primary signal required for the differentiation and fusion of these precursors into mature, multinucleated osteoclasts, which are responsible for bone resorption.

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Figure 3:

Intracellular signaling pathways activated by tumor necrosis factor superfamily member 11 (TNFSF11)/TNFRSF11A binding during osteoclast differentiation and survival. On binding of TNFSF11 (RANKL) to its receptor TNFRSF11A (RANK), the adapter protein tumor necrosis factor receptor-receptor-associated factors (TRAF6) is recruited to the intracellular domain. This recruitment initiates two major signaling cascades: The nuclear factor-kappa B (NF-kB) cascade: Critical for initiating the genetic program of osteoclast differentiation. This leads to the dimerization of NF-kB and its translocation to the nucleus, where it promotes the transcription of key osteoclastogenic genes (e.g., nuclear factor of activated T-cells cytoplasmic 1, TRAF6). The PI3K/Akt Cascade: This pathway, also involving TRAF6, activates Akt. Akt subsequently activates the mTOR pathway, which promotes cell survival, growth, and metabolic changes necessary for the developing osteoclast.

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Furthermore, the TNFSF11/TNFRSF11A interaction induces changes in intracellular calcium (Ca⁺⁺) levels. This, in turn, induces the Ca⁺⁺/Calmodulin pathway and, thus, regulates various osteoclastic activities, including bone resorption.^[76] In addition, mature osteoclasts secrete hydrochloric acid (HCl) and lysosomal enzymes (e.g., cathepsin K) that degrade the mineralized bone matrix, allowing for bone resorption.^[77]

Osteoporosis and over-activation of the TNFSF11/TNFRSF11A pathway

In osteoporosis, the overactivation of the TNFSF11/TNFRSF11A pathway causes excessive osteoclast activity, resulting in increased bone resorption. Factors such as hormonal changes and inflammation can elevate the expression of TNFSF11, disrupting the delicate equilibrium between bone resorption and formation. This imbalance contributes to bone loss and a decrease in bone mineral density, thereby increasing the susceptibility to fractures. Therapeutic targeting of this pathway, such as with denosumab – a monoclonal antibody that specifically binds to TNFSF11 – can effectively treat osteoporosis by inhibiting the TNFSF11/TNFRSF11A interaction, thereby reducing osteoclast activity and bone resorption.^[78] Denosumab, along with other TNFSF11 inhibitors, has been developed to treat diseases with excessive bone resorption. Denosumab prevents the TNFSF11/TNFRSF11A interaction, reducing osteoclast formation and activity. It has been approved for osteoporosis treatment to decrease fracture risk in patients with low bone density.^[79]

Osteopetrosis and impaired TNFSF11/TNFRSF11A pathway

Variations in TNFSF11 or TNFRSF11A can impair osteoclast differentiation and activity, leading to insufficient bone resorption and excessive bone tissue accumulation, which can cause osteopetrosis.^[80]