Bone fragility in patients affected by congenital diseases non skeletal in origin

Several defects can exist in the conversion of the sulfur-containing amino acid methionine to cysteine and the ultimate oxidation of cysteine to inorganic sulfate [18]. The most relevant disorder is rapresented by homocystinuria. Cystathionine-β-synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine (HCY) [18]. It catalyzes the pyridoxal 5′-phosphate (PLP)-dependent’-replacement reaction in which the thiolate of L-homocysteine replaces the hydroxyl group of L-serine [19]. CBS is an especially interesting PLP enzyme because it has a complex domain structure and regulatory mechanism. The allosteric activator, S-adenosyl-L-methionine (AdoMet), increases CBS activity about threefold, and likely binds to the C-terminal regulatory domain [19].

The clinical features of untreated homocystinuria due to CBS deficiency usually manifest in the first or second decade of life and include myopia, ectopia lentis, mental retardation, skeletal anomalies resembling Marfan syndrome (MFS), and thromboembolic events. Light skin and hair can also be present. It is extremely heterogeneous, ranging from patients presenting with all of the complications to individuals with no overt clinical involvement. Biochemical features include increased urinary homocystine and methionine. There are 2 main phenotypes of the classic disorder: a milder pyridoxine (vitamin B6)-responsive form, and a more severe pyridoxine-nonresponsive form. Pyridoxine is a cofactor for the CBS enzyme, and can aid in the conversion of homocysteine to cysteine [20‐22].

Classic homocystinuria is an autosomal recessive metabolic disorder of sulfur metabolism. The human hereditary disease is characterized by very high plasma levels of the toxic amino acid L-homocysteine. The gene coding CBS is localized on chromosome 21q22.3. A large number of mutations in different regions of the human CBS have been found in patients with homocystinuria [19, 23, 24]. Mutations in the CBS gene can alter the mRNA stability, the enzyme activity, the binding of PLP and heme and the allosteric regulation. Rarely, homocystinuria can be caused by mutations in other genes. The enzymes encoded by the MTHFR, MTR, MTRR, and MMADHC genes play a role in converting homocysteine to methionine [23‐25].

Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations, including typical skeletal abnormalities. This aspect of hyperhomocysteinemia has been investigated in the skeleton of cbs-deficient mice, a murine model of severe hyperhomocysteinemia, characterized by impaired cartilage differentiation, albeit to differing degrees [25, 26]. Interestingly, basal levels of cbs gene mRNA and protein in MC3T3-E1 murine pre-osteoblasts appear to increase after incubation with 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] providing evidence of a transcriptional regulation of the vitamin D receptor (VDR) [26], with a possible supporting role for calcitriol on the action of folic acid, vitamin B6, and B12 in lowering high HCY levels [27].

Some of the skeletal abnormalities, are due to the fact that cysteine deficiency, which occurs in hyperhomocysteinemia due to CBS deficiency, can induce a fibrillin-1 defect [28].

Because collagen cross-links are important for the stability and strength of the bone matrix, CBS deficiency patients are prone to fragile bone [29]. In addition, homocysteine levels increase the risk of fractures, independent of bone mineral density (BMD) and other potential risk factors for fracture [29, 30], likely for a disruption of a normal microfibrillar configuration [22].

For early diagnosed patients, treatment can realistically aim to prevent all the complications of CBS deficiency, as normal growth and nutrition, allowing the patient normal opportunities for employment and family life. For late-diagnosed patients, the aim is to prevent further complications, especially thromboembolic disease. Good compliance with low dietary methionine restriction treatment prevented ectopia lentis, osteoporosis and thromboembolic events and it also led to normal cognitive functions. Enzyme replacement therapy based on polyethylene glycol (PEG)ylated 20NHS CBS conjugate represents the most suitable candidate for manufacturing and clinical development [31].

Table 1B describes only homocystinuria phenotype disease as the main disorder of sulfur metabolism, where bone involvement has been described.

Alkaptonuria is an inherited disorder of aromatic amino acid metabolism and results from absence of homogentisate 1,2 dioxygenase (HGD), the enzyme predominantly produced by hepatocytes in the liver and in the kidney, responsible for the breakdown of homogentisic acid (HGA). Deficient HGD activity within the liver causes HGA levels to rise systemically. Large (gram) quantities of HGA are removed by urinary excretion on a daily basis [32, 33].

It is an autosomal recessive genetic disease, meaning that patients have inherited two defective copies of a gene; one from each parent. By 1995, the genetic defect was discovered, cloned, and mapped to chromosome 3 between regions 3q21-q23  [32‐34].

The development of transgenic mice and in vitro models have enabled a better understanding of the pathophysiology involved in the progression of ochronosis and the related osteoarthropathy. Research has identified that HGA is present in healthy cartilage but becomes susceptible to degeneration only following focal changes [35]. Indeed, extracellular matrix (ECM) is normally resistant to ochronosis, but may become susceptible to pigmentation in response to tissue biochemical or mechanical damage, including microtrauma [35]. Pigmentation appears to protect collagen fibers from the action of proteolytic enzymes, contributing to increased tissue stiffness. This is a probable cause of higher susceptibility to matrix damage through normal loading [35]. Pigmentation is shown to begin in the pericellular and territorial matrices of individual chondrocytes and, thus, matrix turnover events in these regions could be an important factor for initiation of pigmentation into the hyaline cartilage [35, 36]. This process of enhanced osteoclastic activity resorbing the unloading subchondral bone and calcified cartilage is similar to the manifestation reported in osteoarthritis [37‐40]. Although aggressive resorption appears to be focal, it is noteworthy that enhanced urinary excretion of crosslinked N-telopeptides of type I collagen has been reported in alkaptonuria patients [34]. In addition, Aliberti et al. [41] showed an increase of bone resorption along with an almost normal bone formation in patients with alkaptonuria, suggesting an enlarged bone remodeling space. In term of bone mass, they found conflicting results about spinal and femoral BMD, as lumbar spine BMD was normal or markedly increased, whilst femoral neck BMD was almost always reduced [41]. It is likely that BMD of lumbar spine might not be reliably assessed in these particular patients. It may be hypothesized that the homogentisic acid polymer deposit in the bone matrix and osteocytes may play a pathophysiological role in accelerating bone loss [41]. It is plausible that in a bone tissue diffusely osteoporotic, the ochronotic pigment is deposited in bone matrix and osteocytes with degenerated or dead cells [41].

In a phase 3 study patients with AKU were given 2 mg of nitisinone each day for three years. Newly published results show that the drug stops the disease, by decreasing HGA. Further, nitisinone therapy not only arrested but also partially reversed ochronosis. Results also show that it significantly reduces the damage caused by ochronosis, especially in the joints. Patients who took nitisinone showed major health benefits [42].

Phenylketonuria (PKU) is a genetic disorder caused by mutations in the gene coding for PAH. As a consequence, the essential amino acid phenylalanine (Phe) cannot be converted to tyrosine and accumulates in the blood.

The human PAH gene, which is located on chromosome 12q, consists of 13 exons spanning 90 kb. To date, more than 520 different mutations in the PAH gene have been characterized in PKU patients and recorded in the PAH Mutation Analysis Consortium Database (https://​www.​pahdb.​mcgill.​ca). Although most of these mutations are detectable by sequence analysis, with a detection rate of 95%, large intragenic deletions or duplications cannot be identified using this method. In these cases, multiplex ligation-dependent probe amplification (MLPA) can be used as a sensitive and efficient method [43, 44].

Bone complications are seen in early and continuously treated patients [44]. Indeed, most studies on patients with PKU indicate that bone is often affected. However, there are significant gaps in the pathogenetic basis of this complication neither consensus exists on the degree and implications of bone abnormalities and the risk factors for low BMD [4, 5, 14]. To investigate these gaps, a meta-analysis on BMD, corrected for bias, age and gender, has been performed by Rucker RB et al. [45]. The Authors found that BMD in early diagnosed and treated patients with PKU is below the healthy population average but within the normal range [45]. Moreover, data on a corresponding higher risk of fracture in these patients are missing [45].

Other indicators of bone status in early treated patients with PKU are inconclusive due to the small number of studies and the heterogeneity in the groups examined and in the measurement methods. A recent meta-analysis found that low BMD does not seem to be an exaggerated concern in patients with PKU, and that research is needed on the effects of the PKU diet on bone, on the reliability of bone turnover markers in bone assessment, and on a concrete estimate of fracture risk in patients with PKU [45].

Dietary treatment is the basis of PKU management. It consists of 3 components: natural protein restriction, Phe-free-L-amino acid supplements, and low protein food intake. Effectiveness of PKU treatment is demonstrated by any of the following objective measures: reduction in Phe blood concentrations, increase in natural protein tolerance, improvement in neuropsychological testing, in the nutritional status, and in the quality of life [46]. A possible enzyme replacement therapy using PEG Phenylalanine-Ammonia Lyase (PAL) or Pegvaliase is under investigation [46]. PEG-PAL trials have proven short-term reduction in the Phe blood concentrations in adult PKU patients, but further studies are required to observe long-term effectiveness and safety. Results of a phase III extension study (NCT01819727) are awaited. Gene therapy and therapeutic liver repopulation have been investigated in murine models only, and larger animal PKU models and human studies need to be developed [46].

Table 1C describes the diseases due to the alteration of tyrosine pathway in which the skeletal involvement has been described.

The physiological importance of Cu-ATPases in humans can be illustrated by the deleterious consequences of the Cu-ATPase inactivation on cell metabolism. Mutations or deletions in the ATP7A gene encoding the Cu-ATPase ATP7A are associated with a fatal childhood disorder, Menkes disease. The Menkes gene is located on the long arm of the X chromosome at Xq13.3, and the gene product, ATP7A, is a 1500–amino acid P-type adenosine triphosphatase (ATPase) that has 17 domains—6 copper binding, 8 transmembrane, a phosphatase, a phosphorylation, and an ATP binding. The lack of functional ATP7A is associated with severe connective tissue defects in Menkes disease patients, likely due to disrupted delivery of Cu to lysyl oxidase in the secretory pathway. The connective tissue defects, including vascular tortuosity, loose skin, hyperextensible joints and bone fragility, are commonly observed in Menkes disease [48].

Cu has an essential role in the normal maturation of collagen, particularly in the important steps to the formation of lysine-derived cross-link skeletal findings in infants with Menkes disease, the most characteristic of which are metaphyseal spurs, long-bone fractures, and wormian bones [52‐54]. The long-term findings have been described by Amador et al. [52] and differ completely from those initially observed. In particular, undertubulation and metaphyseal flaring, similar to the findings seen in some types of bone dysplasia, are present [52]. In addition, the defective collagen cross-linking leads to osteoporosis and pathological fractures in these children [53, 54].

Mutations in the gene encoding the Cu-ATPase ATP7B also result in a severe metabolic disorder, known as Wilson disease (WD), which is inherited in an autosomal recessive manner. The ATP7B gene is approximately 80 Kb, located on human chromosome 13, and consists of 21 exons. The mRNA transcribed by the ATP7B gene encodes a protein of 1465 amino acids [57]. This protein is part of the P-type ATPase family, a group of proteins that transports metals into and out of cells by using energy stored in the molecule adenosine triphosphate (ATP). Cu-transporting ATPase 2 is found primarily in the liver, with smaller amounts in the kidneys and in the brain. WD is caused by various mutations in the ATP7B gene. In Western populations, the H1069Q mutation is present in 37–63% of cases, while in China this mutation is very uncommon and R778L is found more often. Ten percent of patients have no detectable mutations in the ATP7B gene [58].

Skeletal changes have been reported in WD, including osteoporosis [59, 60]. It is typically associated with an accumulation of Cu in various tissues, including bone tissue, with skeletal Cu content reported to be increased by about four times [60]. In a study by Hegedus et al., beta-cross-laps, a marker of bone remodeling, were found to be significantly increased in WD patients, suggesting increased bone resorption as a key mechanism contributing to bone loss and osteoporosis in these patients [61]. Weiss et al. [59] showed that the severity of liver disease is unrelated to the degree of bone loss as assessed by DXA, hypothesizing that Cu accumulation may directly affect bone metabolism. Indeed, accumulation of Cu causes production of reactive oxygen species (ROS) which is a risk factor for osteoporosis and may accelerate the effect of aging of bone by stimulating osteoclast differentiation and bone resorption and inhibiting osteoblast differentiation [59, 62]. In addition, an excess of Cu is able to induce mitochondrial dysfunction and mitochondrial toxicity, which are causally related to bone demineralization [59].

In guidelines for the American Association for the Study of Liver Diseases, medical therapy is divided into 2 categories: treatment of symptomatic patients and treatment of asymptomatic patients [63]. Treatment of symptomatic patients (hepatic, neurologic, or psychiatric) should include chelation therapy, either with D-penicillamine or with trientine that increases urinary Cu excretion. Patients with WD are counseled to eat a diet low in Cu and avoid the very high Cu foods, such as mushrooms, chocolate, liver, and nuts [63]. Studies with sodium tetrathiomolybdate for the treatment of WD suggested some benefit for its use as initial treatment of patients with neurological complications [63]. A phase 2 study was conducted with a more stable form of a chelator for Cu, the choline tetrathiomolybdate (WTX101) molecule, that has the potential to increase biliary Cu excretion as well as bind Cu and albumin in an inert complex [64, 65]. Liver transplant can be used to rescue those with acute liver failure or those with liver disease who fail to respond to medical therapy. New advances in treatment may improve adherence and reduce some complications of treatment [64, 65].

Table 2 describes the liver diseases due to the alterations in the Cu metabolic pathway.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis [68]. The CFTR gene is localized on chromosome 7q31.2 and provides instructions for making a channel that transports negatively charged particles called chloride (Cl) ions into and out of the cells [69].

Cl is a component of sodium chloride, a common salt found in sweat, but it also has important functions in cellular metabolism, as the flow of Cl ions helps to control the movement of water in tissues [69]. Mutations in the CF transmembrane conductance regulator (CFTR) gene disrupt the function of the Cl channels, preventing them from regulating the flow of Cl ions and water across cell membranes. As a result, living cells in lungs, pancreas and other organs produce mucus that is unusually thick and sticky [69‐71].

Major pathogenic mechanisms mediating the development of CF bone disease (CFBD) in patients with CF may result from a combination of episodes of low bone turnover and formation rate during periods of disease stability, and an increased bone turnover and resorption during infective exacerbations [72]. Osteoporosis and increased vertebral fracture risk have been associated with CF disease, becoming more relevant with the increase in life expectancy of these patients. The first report on association of low BMD with CF was published in 1979 [71]. The etiology of low bone density is multifactorial, most probably a combination of inadequate peak bone mass during puberty and increased bone loss in adults [72]. Body mass index, male sex, advanced pulmonary disease, malnutrition and chronic therapies are established additional risk factors for CFBD [72]. Fractures not only cause pain and disability, but vertebral fractures and kyphosis further impair breathing biomechanics and often disqualify a patient from lung transplantation [73, 74]. Stalvey et al. developed and showed a CF disease model in which CFTR expression in bone directs reduced osteoblast differentiation and enhanced osteoclastic bone resorption [73]. This report, and other studies, demonstrate that CFTR is expressed in mesenchyme-derived osteoblasts, odontoblasts, chondrocytes and myocytes, and that mutations directly perturb cellular activities of these cell types [75, 76]. The in vitro evidence of delayed osteoblast new bone formation, and enhanced osteoclastic bone resorption resulting from reduced osteoprotegerin expression parallels the uncoupled bone turnover that is characteristic of CFBD in humans and in animal models [77, 78]. In addition, in human osteoblasts obtained from a 25-year-old CF male with the F508del/G542X mutation in the CFTR gene, a defective CFTR-mediated Cl-channel activity and a severe deficit in the production of osteoprotegerin  was described [77, 78]. These data raised the hypothesis that the Cftr gene may play a role in cell differentiation and bone matrix maturation [79].

The goals of treatment primarily include: 1. preventing and controlling lung infections; 2. controlling of airway inflammation; 3. reducing viscoelasticity by removing thick, sticky mucus from the lungs and dilating the airways A new group of drugs called CFTR modulators are available which are able to correct the basic defect in CF, with an improvement in the management of the disease [75, 80]. However, most of these drugs are having serious hepatic and extra-hepatic side effects, in addition to high costs. Gene engineering techniques and new molecular targets may be explored besides CFTR. With the help of modern biology approaches like DNA nanotechnology, systems biology, metabolomics, disease modeling, and intracellular protein kinetics new pathways and networks associated with CF can be unraveled and eventually new therapeutic targets can become available [80, 81].

Table 3 describes the phenotypic characteristics of cystic fibrosis.

Mastocytosis is a complex disorder characterized by the accumulation of abnormal mast cells (MCs) in the skin, bone marrow and/or other visceral organs. The clinical manifestations result from MC-derived mediators and, less frequently, from destructive infiltration of MCs. Patients suffer from a variety of symptoms, including pruritus, flushing and life-threatening anaphylaxis. While mastocytosis is likely to be suspected in a patient with typical skin lesions (i.e. urticaria pigmentosa [UP]), the absence of cutaneous signs does not rule out the diagnosis of this disease. Mastocytosis should be suspected in cases of recurrent, unexplained or severe insect-induced anaphylaxis or symptoms of MC degranulation without true allergy [84].

In rare cases, unexplained osteoporosis or unexplained hematological abnormalities can be the underlying features of mastocytosis, particularly when these conditions are associated with elevated baseline serum tryptase levels. The diagnosis is based on the World Health Organization (WHO) criteria, in which serum tryptase levels, histopathological and immunophenotypic evaluation of MCs and molecular analysis are crucial [85]. The WHO classification subdivides mastocytosis into seven major categories: (1) cutaneous mastocytosis, (2) indolent systemic mastocytosis, (3) systemic mastocytosis with associated clonal, hematological non-mast-cell lineage disease, (4) aggressive systemic mastocytosis, (5) mast cell leukemia, (6) mast cell sarcoma, and (7) extracutaneous mastocytoma. Within these major categories additional variants and subvariants coexist, some of which are currently considered ‘provisional entities’ [85].

Mastocytosis is associated with a somatic gain of function point mutation in the KIT gene. This was firstly recognized in 1995, when Nagata et al. [86] identified a point mutation in exon 17, resulting in a substitution of valine for aspartic acid in the catalytic domain of the KIT gene (D816V) in the peripheral blood of four patients with mastocytosis. Further analysis of larger cohorts confirmed that the KIT gene D816V mutationis detected in up to 93% of adult patients with the disease [84]. By contrast, patients with childhood onset mastocytosis may not have detectable KIT mutations or may express KIT mutations other than D816V (in exons 8, 9, 11 or 17) [84].

Osteopenia/osteoporosis with bone pain and possible pathological fractures has long been recognized in systemic mastocytosis (SM) patients. Half of adult patients with SM show bone involvement. Osteoporosis is the most prevalent bone manifestation in SM (31%) [87]. Serum Interleukin 6 (IL-6) is elevated in mastocytosis patients and correlates with severity of symptoms and the presence of osteoporosis. High serum IL-6 may not only signify disease progression, but may also participate in the pathophysiology of SM. The pathological fractures are considered a marker of aggressive SM, especially when associated with high levels of serum tryptase [84]. The main actor is the osteoclast with a relative or absolute predominance of bone resorption versus formation. Among the stimuli that drive osteoclast activity, the most important is the Receptor Activator of Nuclear Factor κ B (RANK)—RANK-Ligand (RANKL) signaling, but also histamine and other cytokines play a significant role in the process [84].

During the last two decades, major discoveries contributed to a better diagnosis, identification of the clinical and biological abnormalities, and a refined classication of the different forms of mast cell disease [84]. Various cytoreductive treatments have been used for advanced SM, including 2-chlorodeoxyadenosine (2-CDA), interferon-alpha (IFN-α), classical chemotherapy agents (such as cytarabine or udarabine), but all with modest and disappointing response rates, highlighting the need for innovative therapies [84]. Tyrosine kinase inhibitors (TKIs) are an attractive therapeutic approach, given the pathogenesis of SM and the involvement of KITD816V mutation in more than 80% of patients, and other KIT mutations that map to the TK juxtamembrane domain or transmembrane domain in sporadic cases of SM. Many studies have reported quantitative and qualitative defects of signal transduction in SM [88]. These altered pathways play a role in the pathogenesis of SM and targeted drugs may provide therapeutic options by selective inhibition of some of these critical targets. Finally, normal and neoplastic mast cells express on their surface a number of cell surface antigens that might be considered as potential targeted therapies in advanced SM [88]. The central role of osteoclasts made bisphosphonates, as anti-resorptive drugs, the standard of treatment for bone involvement in SM [89]. Bisphosphonate therapy seems to improve lumbar spine BMD during SM-related osteoporosis. Spine x-ray and BMD should be performed in all SM patients to detect those who may benefit from an osteoporosis therapy [89].

Thalassemia is an inherited blood disorder characterized by a defect in the globin chain synthesis in red blood cells. The clinical phenotype results from both the diminished amount of the particular globin chain as well as from the resultant chain imbalance that occurs because of normal production of the other globin chain [90]. The common forms include alpha (α) and beta (β) thalassemia [90]. In β thalassemia, the synthesis of normal α globin chains from the unaffected α globin genes continues as normal, resulting in the accumulation within the erythroid precursors of excess unmatched α globin [91]. In α thalassemia many mutations can affect the α globin gene, but the most common are gene deletions [91]. Anemia in β thalassemia results from a combination of ineffective erythropoiesis, peripheral hemolysis, and an overall reduction in hemoglobin synthesis. The severity of disease in β thalassemia correlates well with the degree of imbalance between α and non-α globin chains and the size of the free α chain pool. Thus, factors that reduce the degree of chain imbalance and the magnitude of α chain excess in the red cell precursors will have an impact on the final phenotype [91]. It might be important to note that the clinical phenotype becomes most apparent at 6–9 months of age, due to the fetal to adult hemoglobin switch that occurs at that age [90]. Finally, β thalassemia is the only form of this disorder where bone metabolism is involved.

Thalassemia results when mutations affecting the genes involved in hemoglobin (Hb) biosynthesis lead to decreased Hb production. Depending on the mutation of genes located on chromosome 16 and 11 we can distinguish α and β thalassemia, respectively [90]. Mutations in the HBB gene cause β thalassemia. The HBB gene provides instructions for making beta-globin a subunit of hemoglobin. Some mutations in the HBB gene prevent the production of β globin [91]. About 100 mutations have been described that decrease β chain synthesis. Mutations fall into two classes: B0 refers to mutations that cause no β globin to be produced and B+ describes mutations that result in a diminished but not absent quantity of β globulin. The severity of these mutations can vary depending on the amount of normal β globin that is produced [91]. Depending the class of mutation present and the gene dosage (i.e. heterozygous or homozygous), patients can present with different severity of the disease [90]: (a) β thalassemia major (TM) refers to a severe clinical phenotype; (b) β thalassemia intermedia is characterized by a clinical phenotype based on heterogenous genetic mutations that still allow for some β chain production (e.g. B + /B0, B + /B +); and (c) β thalassemia minor/ thalassemia trait shows a mild clinical phenotype with a normal copy of the β globin gene being present (e.g. B + /B, B0/B) [90].

Metabolic bone disease represents a major cause of morbidity in patients with TM (the most severe form). The pathogenic factors include: the primary disease itself that causes bone marrow expansion, the chronic anemia, and the iron endocrine toxicity [92, 93].

Marrow expansion causes mechanical interruption of bone formation, leading to cortical thinning and increased distortion and fragility of the bone [93]. Other factors lead to alterations in the RANK/RANKL/osteoprotegerin system, increasing osteoclastic activity and enhancing osteoblast dysfunction [94, 95]. The increase of RANKL, followed by unmodified osteoprotegerin levels, with the consequent increase of RANKL/osteoprotegerin ratio, may represent the cause of the uncoupling of bone turnover observed in thalassemia patients. However, no correlation between sRANKL or osteoprotegerin levels with BMD of the lumbar spine or at the femoral neck is present [93].

Hypogonadotrophic hypogonadism and delayed puberty are the most common endocrine complications in patients with TM, and they also contribute to osteopenia and osteoporosis. Morabito et al. [96] showed a negative correlation between RANKL and free testosterone in male thalassemia patients and with 17β-estradiol in female thalassemia patients, which suggests that a reduced production of sex steroids causes an increase in RANKL production [96]. There are gender differences not only in the prevalence but also in the severity of the osteoporosis syndrome. The reported frequency of osteoporosis, even in well-treated TM patients, varies from 13.6% to 50% with an additional 45% affected by osteopenia [92]. The lack of an anabolic effects of growth hormone (GH) and insulin-growth factor I (IGF-1) on bone formation for the acquisition of bone mass, mainly during childhood and puberty, is clearly involved in the pathogenesis is of bone mass reduction in thalassemic patients.

Transfusion therapy works by supplying normal erythrocytes and suppressing ineffective erythropoiesis, essentially controlling all downstream pathophysiological mechanisms in thalassemia. Whether iron overload develops from increased intestinal absorption or secondary to regular transfusions, it can cause substantial morbidity and mortality, and so warrants prompt diagnosis and effective management. For this reason it is important to use iron chelators in the affected patients. Three iron chelators are currently available for the treatment of iron overload in patients with thalassemia: deferoxamine, deferiprone and deferasirox. Finally, splenectomies have traditionally been performed as an alternate or adjunct therapy to transfusion [93]. In TM, GH secretory dysfunction is common and contributes to osteopenia and osteoporosis, along with other endocrinopathies, such as hypoparathyroidism, vitamin D deficiency, hypothyroidism, and diabetes [94]. Prevention is without a doubt the first step in the management of osteoporosis in TM, with the final goal of preventing bone fragility and fractures. The management of patients with TM should start as early as at birth in order to minimize disease complications [85‐94].

Figure 3 shows the mechanisms responsible for the pathogenesis of thalassemia.

Hemophilia is a male bleeding disorder with worldwide distribution. Affected individuals have either reduced or absent levels of coagulation factor VIII and IX in plasma (hemophilia A and hemophilia B respectively). Hence, they encounter unusual bleeding, either spontaneous or after trauma and surgery. The spectrum of bleeding manifestations varies from superficial ecchymosis to lethal hemorrhage in the central nervous system [97, 98]. While the prevalence of hemophilia A varies among different countries, its prevalence has been estimated to be about 3 to 20 per 100,000 in the population. Hemophilia B is seen as 1 case among 5,000 male births in European countries and the United States [97, 98]. Individuals with hemophilia A and B have respectively either reduced or absent levels of coagulation factor VIII and IX. Lack or reduced levels of coagulation factors VIII and IX result in impaired clot formation and tendency to bleeding episodes. These lifelong disorders are categorized as severe type (< 1%), medium type (1–5%), and mild type (5–30%)  [98]. Replacement therapy using plasma derived or recombinant coagulation factor concentrates are the basis of treatment for bleeding episodes and prophylaxis regimens [99].

A X-linked, recessive hemorrhagic trait or gene induces Hemophilia A. Hemophilia A X-linked trait manifests as a congenital absence or decrease in plasma clotting Factor VIII, a pro-coagulation cofactor and robust initiator of thrombin that is essential for the generation of adequate amounts of fibrin to form a platelet/fibrin plug at sites of endothelial disruption. Female hemophilia gene carriers do not manifest symptoms of Hemophilia A but may have lower than usual quantities of Factor VIII. Male Hemophilia A patients do not transmit hemophilia to male offspring, but all their female offspring will carry the hemophilia gene.

Twenty-seven percent of hemophilic patients have osteoporosis and 43% osteopenia [92]. A large and growing body of literature has investigated osteopenia and osteoporosis both in children and in adults with hemophilia [98, 100, 101]. While physical activity is considered as a central indicator of bone resorption, patients with bleeding disorders are less likely to engage in high impact activities and weight-bearing exercises [98]. . Hemarthrosis, infection with hepatitis C virus are also commonly reported causes of susceptibility of hemophilia to reduced bone density [98]. Recently, a survey in hemophilia A, hemophilia B and von Willebrand disease (vWD) showed a higher risk of bone health outcomes in comparison with healthly controls and suggested a possible role of endogenous coagulation factors in the maintainanceof bone mass [102].

The first successful treatment of hemophilia A with whole blood transfusion was reported in 1840 and subsequently, treatment with plasma was introduced,  with the cryoprecipitate fractions of plasma enriched in FVIII firstly utilized in 1964 [97]. High purity Factor VIII concentrate treatment started in 1984 with the production of recombinant FVIII (rFVIII) generated through cloning of FVIII [103]. The development of recombinant FVIII (rFVIII) infusion has improved the life expectancy of patients with mild to moderate hemophilia A, reaching levels comparable to that of the general population. However, an ongoing concern is the development of inhibitory antibodies to plasma-derived FVIII or rFVIII. Prophylaxis appears to be used less frequently in hemophilia B than in hemophilia A patients.

Transplantation and engraftment of liver sinusoidal endothelial cells (LSECs) capable of producing FVIII provides the most natural pathway for the cure of hemophilia A. Such a cell based therapy for hemophilia A offers the potential of a life-long cure if a number of hurdles can be overcome. Hemophilia B may have been relatively neglected compared to haemophilia A, but of note, major recent advances in haemophilia therapy (i.e. longer-acting factor concentrates with extended half-lives, and gene therapy) will potentially be available for haemophilia B before haemophilia A [104, 105]. For bone health, the results of a study on the effect of prophylaxis in hemophilia indicated that replacement therapy as a prophylaxis regimen beginning in early childhood may preserve normal BMD in severe hemophilia patients [106].

Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb) synthesis affecting many individuals throughout the world. SCD manifests itself soon after the protective effect of HbF diminishes. Its complications vary: acute chest syndrome, proliferative retinopathy, pulmonary hypertension, renal insufficiency, cerebral vascular accident, and musculoskeletal complications [107]. SCD is classified by the number of globin genes that are affected and inherited in an autosomal recessive manner. The heterozygous form is when the individual only carries one gene for HbS and is called sickle cell trait. It occurs frequently in individuals of African ancestry with 8% to 10% of African Americans carrying the trait. This is benign and the affected individual will not show any symptoms and no treatment or precautions are needed. The homozygous state is the most common and severe form of SCD and is called sickle cell anemia (HbSS), accounting for 60–70% of SCD in the United States (US). In HbSS, the red blood cells are distorted and sickle shaped [107]. The two major characteristics are chronic hemolytic anemia and intermittent vaso-occlusion that results in tissue ischemia and causes acute, severe pain episodes. SCD is a chronic disease that has detrimental effects on the entire body [107].

SCD encompasses a group of disorders characterized by the presence of at least one hemoglobin S allele (HbS; p.Glu6Val in HBB) and a second HBB pathogenic variant resulting in abnormal hemoglobin polymerization. HbS/S (homozygous p.Glu6Val in HBB) accounts for 60–70% of SCD in the United States. Other forms of SCD result from coinheritance of HbS with other abnormal βglobin chain variants. SCD is inherited in an autosomal recessive manner. If one parent is a carrier of the HBB HbS pathogenic gene variant and the other is a carrier of any of the HBB pathogenic variants (e.g., HbS, HbC, β thalassemia), each child has a 25% chance of being affected, a 50% chance of being an unaffected carrier, and a 25% chance of being unaffected and not a carrier. Prenatal diagnosis for pregnancies at increased risk for SCD is possible by molecular genetic testing if the HBB pathogenic variants have been identified in the parents [107].

The bone involvement in SCD ranges from acute manifestations, such as painful vaso-occlusive crisis or osteomyelitis, to more chronic and debilitating complications, such as osteonecrosis, osteoporosis and osteopenia, impaired growth and chronic infections [107]. Although these bone complications may not contribute directly to mortality, they are the major source of morbidity with a high impact on patients’ quality of life [107]. Osteopenia and osteoporosis are often asymptomatic but may cause pain, fractures, deformity, and vertebral collapse [107‐109].

Serrai et al. [110] observed a high prevalence (76%) of abnormal BMD (low and very low) by DXA in SCD adults, with predilection for the lumbar spine, and they found a strong correlation between low Hb level and abnormal BMD [110]. Chronic and severe anemia places a burden on the bone marrow, with increased erythropoiesis causing hyperplasia of the bone marrow, decrease in the trabecular network, osteopenia and bone destruction [110]. In addition, various micronutrient and macronutrient deficiencies leading to delayed skeletal maturation (malnutrition, vitamin D and zinc deficiency, etc.), hormonal deficiency (reduced GH, hypogonadism), and poor weight bearing because of chronic pain and inactivity, may all contribute to the development of sickle bone disease [108, 109]. Over 70% of adults with SCD will develop low BMD, which typically occurs 2–3 decades earlier (median age 30 years) than in the general population [108].

Hematopoietic stem cell transplantation has the potential to establish normal erythropoiesis with stable long-term engraftment, amelioration of symptoms, and stabilization of organ damage. Early results in gene therapy for SCD are encouraging. Evaluation of the long-term benefits of curative therapies for SCD requires comparative clinical trials and studies of late effects of transplantation [111].

Ghosal hematodiaphyseal dysplasia is a rare inherited condition characterized by abnormally thick bones and a shortage of red blood cells [112]. It is an autosomal recessive disorder characterized by metadiaphyseal dysplasia of the long bones and defective hematopoiesis due to fibrosis or sclerosis of the bone marrow [112]. The disease was described for the first time by SP Ghosal in five children who had moderate to marked anemia and diaphyseal dysplasia [112]. Hematologic manifestations of the syndrome include varying degrees of anemia, thrombocytopenia, or pancytopenia [112]. Only a few cases have been reported in the medical literature. Most affected individuals have been from the Middle East and India. Treatment consists of steroid therapy and most cases reported in literature previously required a low-dose maintenance therapy throughout life to keep hemoglobin at normal levels [112].

Ghosal hematodiaphyseal dysplasia results from mutations in the thromboxane synthase TBXAS1 gene localized on chromosome 7q34. This gene encodes thromboxane-A-synthase, an enzyme which produces thromboxane A2(TA2). TA2 is responsible for platelet aggregation (via the arachidonic acid cascade) and also modulates bone mineral density variation (by influencing the expression of TNFSF11 and TNFRSF11B, which encode RANKL and osteoprotegerin in osteoblasts, leading to bone sclerosis). Most reported mutations in the TBXAS1 gene are of missense type and the pathogenesis of the bony and hematological abnormalities are presumed to be due to a sclerosing bone dysplasia secondarily leading to marrow hypocellularity [112].

In a Caucasian patient with two biallelic mutations within the gene TBXAS1 (c.266 T > C; c.989 T > C) bone densitometry revealed increased bone density with Z-score of 4.5, consistent with mild thickening of long bone diaphysis noted via X-ray [113, 114]. Strikingly, the bone marrow biopsy revealed haphazard ossification, immature cartilage formation and thickened trabeculae replacing hematopoiesis within the marrow cavity [112, 113]. TBXAS1 defects decrease TXA2-mediated osteoblast RANKL expression paralleled by elevated production of osteoprotegerin, ultimately inhibiting osteoclast differentiation [113]. In addition, increased PGE2 synthesis may induce inflammation and increase bone density [113]. Similar to the Ghosal type hemato-diaphyseal dysplasia is the Camurati-Engelmann disease, an extremely rare disease characterized by hyperostosis of multiple long bones caused by mutations in the TGFB1 gene. However, in the Camurati-Engelmann disease, only the diaphysis is affected, while both diaphysis and metaphysis are affected in Ghosal type hemato-diaphyseal dysplasia [83].

No specific therapy exists.

Demographics play an important part in epidemiology. Worldwide, autosomal dominant disorders seem to be more common, whereas the recessive disorders are usually diagnosed in consanguineous kindreds [115]. Several genes can be involved in severe congenital neutropenia and play a role in the maturation and function of neutrophils, promoting cell survival and response to immune signals. Most patients (about 50%) harbor mutations of the ELANE gene [83], encoding the neutrophil elastase, while about 10% of patients instead harbor mutations in the HAX1 gene, encoding the HS-1-associated protein X-1 (HAX1), where HS-1 is a Src family tyrosine kinase substrate and HAX1 is a mitochondrial protein that regulates apoptosis [83]. HAX1 protein contributes to the activation of the granulocyte colony-stimulating factor (G-CSF) signaling pathway and could cause osteopenia enhancing bone resorption [83]. Various other genes, including CSF3R, G6PC3, GFI1, JAGN1, TCIRG1, VPS45 and WAS, account for a small portion of patients, while about 30% have unknown genetic defects, with some cases with no familial history, thus classified as sporadic [117]. The GFI1 gene encodes a transcription factor that controls human stem cells quiescence and osteoblast differentiation, inducing epigenetic changes in the RUNX2 promoter [83].

SCN is associated with osteopenia in at least 40% of patients, which can then evolve towards overt osteoporosis. Recurrent infections and diminished physical activity may play a prominent role in decreasing BMD because of increased catabolism and poor nutritional status [116]. On the other hand, exogenous administration and genetically induced overexpression of G-CSF have been related to bone mineral loss in animal studies [116]. On the basis of the evidence that osteoclasts are derived from the monocyte/macrophage lineage, various hypotheses state that a large group of cytokines and CSFs involved in hematopoiesis, including G-CSF, regulate osteoclast development and activity [108]. The receptor activator of nuclear factor-kB (RANK), the RANK-L, and osteoprotegerin. are important regulators of osteoclastogenesis [116]. Elhasid et al. [118] described a 4-year-old patient who had familial SCN and severe osteoporosis with multiple fractures that presented very low levels of osteoprotegerin with a high RANKL/osteoprotegerin ratio. This may play an important role in the pathogenesis of osteoporosis in these patients [118].

No specific therapy is available. The anemia can be treated with oral prednisolone with walking difficulties improvement [86]. Bisphosphonates have been used for the treatment of bone loss [116].

Histiocytoses are a diverse group of hematologic disorders defined by the pathologic infiltration of normal tissues by cells of the mononuclear phagocyte system (MPS). Cells of the MPS have a wide range of morphologic, anatomic and functional characteristics that make classification of this system difficult. The development and differentiation of the cells of the MPS, much like other hematopoietic lineage, is driven by a tightly regulated pattern of gene expression governed by distinctive sets of transcription factors that control cell proliferation and differentiation. As proposed in 1987 [119], histiocytic disorders can be classified into three classes based on the pathologic cells present within the lesions:

This system was revised in 1997 by the WHO Committee on Histiocytic/Reticulum Cell Proliferations and the Reclassification Working Group of the Histiocyte Society [120]. The central theme of this reclassification effort consisted of distinguishing the clearly malignant histiocytoses from the remaining subtypes, the so-called “disorders of varied biological behavior” [121].

Somatic mutations in the BRAF gene have been identified in the Langerhans cells of about half of individuals with Langerhans cell histiocytosis [119]. Somatic gene mutations are acquired during a person's lifetime and are present only in certain cells. These changes are not inherited. A major breakthrough in the LCH puzzle came with discovery of recurrent somatic BRAF V600E gene mutations in histiocytes in 50% of LCH lesions, 13 of which have been validated in multiple subsequent studies [122]. BRAF is a central kinase of the MAPK pathway (RAS/RAF/MEK/ERK) that transduces extra-cellular signals and regulates critical cellular functions [122]. The V600E mutation results in RAS-independent constitutive activation of downstream MEK and ERK [118]. Besides the V600E mutation, single case reports have described additional somatic mutations within the BRAF gene locus (BRAF V600D, BRAF 600DLAT), as well as the germline mutation/polymorphism BRAF T599A with potential functional consequences [122].

The skeletal system is the most common site of involvement of Langerhans cell histiocytosis, and in 60–80% of cases is the only organ system involved. The lesions may be asymptomatic and discovered as an incidental radiographic finding, when symptomatic patients complain pain, swelling and tenderness around the lesion. Systemic symptoms may also be present, including general malaise and, on occasion, fever with leukocytosis [121, 123]. Makras et al. [124] in a retrospective study examined whether BMD and indices of bone turnover are affected in adult patients with LCH and whether these patients are at high risk of developing osteoporotic fractures. Their findings are particularly relevant as such patients are probably at high risk of developing osteoporosis due to the secretion of bone resorbing cytokines from the pathological process and the presence of a number of several predisposing factors such as anterior pituitary deficiencies and treatment with glucocorticoids and/or chemotherapy. Indeed, adults with LCH have high serum osteoprotegerin levels and low serum RANKL levels than controls [125]. In addition, RANKL is abundantly expressed in cells of adult LCH lesions from different tissues, especially within inflammatory infiltrates [125] and concomitant nuclear staining of p65 NFκB, is associated with increased RANKL expression, suggesting that RANKL could be directly involved in any lesional cell activation [125].

LCH has a wide spectrum of clinical presentations and variable outcomes. With current risk-adapted treatments, more than 80% of patients are cured. However, 30% to 50% of patients experience disease recurrence and a significant number of survivors develop neurodegeneration. Treatments aimed at achieving long-term cures with low reactivation rates and that could prevent the development of neurodegeneration need to be developed. Targeted therapies with BRAF or MEK inhibitors offer the possibility of reframing the treatment of LCH in the near future [126]. Denosumab administration, a RANKL inhibitor, seems a rational treatment strategy in LCH in order to enhance further endogenous osteoprotegerin action and interrupt the lesional immunological process if RANKL related. Makras P et al. showed that the administration of denosumab in patients affected by adult LCH resulted in a significant remission of the disease activity at both osseous and nonosseous lesions [127]. The Authors concluded that denosumab could be considered an effective treatment option in adults with multisystem LCH, also exerting a significant analgesic effect on bone lesions [127, 128].

The most frequently affected bones are the femur, the tibia and the fibula and, less frequently, the ulna, the radius and the humerus. Bone pain usually manifests around the knees and ankles. Osteosclerosis occurs bilaterally and symmetrically in the diametaphyseal regions of the long bones. While the classical hallmark of the skeletal involvement of ECD is osteosclerosis, mixed sclerotic and lytic lesions have occasionally been described. Conversely, bone lesions found in LCH are lytic rather than sclerotic [133] and this complicates the diagnostic  iter. The distribution of sclerotic lesions in ECD differs from that of LCH in that the latter commonly involves the calvarium, facial bones, proximal limbs, pelvis, and scapula, rather than the distal limbs as in ECD [133].

For decades, corticosteroids, cytotoxic agents such as vinca alkaloids and immunosuppressive agents (i.e. cyclophosphamide, methotrexate and azathioprine) represented the therapeutic milestone for ECD patients. More recently, interferon (IFN)-based therapy has emerged as a reliable option for ECD patients. Inhibition of BRAF activation by vemurafenib is a highly promising treatment, in particular for its ability to overcome the blood brain barrier. Mutations of BRAF gene result in a conformational change of a serine/threonine-protein kinase that leads to a chronic activation of the RAS-RAF-MEK-ERK pathway, therefore accelerating the proliferation of the cells [133]. Intriguingly, the recent discovery of additional NRAS gene mutations in ECD strongly suggests that the entire RAS-RAF-MEK-ERK pathway plays a pathogenic role in histiocytosis, thus raising interest in dual BRAF/MEK inhibition [134]. Improved understanding of the pathogenetic mechanisms underlying ECD will lead to the development of more effective targeted therapies, while an accurate stratification of patients based on their neurologic involvement will help clinicians to maximize the benefits of novel molecular targeting for therapies.

Table 4 describes the hematological disorders with bone involvement.