Skeletal and Bone Mineral Density Features, Genetic Profile in Congenital Disorders of Glycosylation: Review

Congenital disorders of glycosylation (CDGs) are a heterogeneous group of disorders with impaired glycosylation of proteins and lipids. These conditions have multisystemic clinical manifestations, resulting in gradually progressive complications including skeletal involvement and reduced bone mineral density. Contrary to PMM2-CDG, all remaining CDG, including ALG12-CDG, ALG3-CDG, ALG9-CDG, ALG6-CDG, PGM3-CDG, CSGALNACT1-CDG, SLC35D1-CDG and TMEM-165, are characterized by well-defined skeletal dysplasia. In some of them, prenatal-onset severe skeletal dysplasia is observed associated with early death. Osteoporosis or osteopenia are frequently observed in all CDG types and are more pronounced in adults. Hormonal dysfunction, limited mobility and inadequate diet are common risk factors for reduced bone mineral density. Skeletal involvement in CDGs is underestimated and, thus, should always be carefully investigated and managed to prevent fractures and chronic pain. With the advent of new therapeutic developments for CDGs, the severity of skeletal complications may be reduced. This review focuses on possible mechanisms of skeletal manifestations, risk factors for osteoporosis, and bone markers in reported paediatric and adult CDG patients.


Introduction
Congenital Disorders of Glycosylation (CDGs) are a clinically heterogenous group of over 150 diseases caused by defects in different steps of glycan metabolism pathways [1,2]. These genetic disorders are characterized by an impaired synthesis and attachment of glycans to glycoproteins and glycolipids, and impaired synthesis of glycosylphosphatidylinositol (GPI) (Figure 1) [1]. These disorders are categorized into (a) protein N-glycosylation defects; (b) protein O-glycosylation defects; (c) glycolipid and GPI anchor synthesis defects and (d) multiple glycosylation pathways and other pathways defects [2]. Among the different glycosylation defects, impaired protein N-glycosylation is the most common. Glycan processing (remodeling) takes place in the endoplasmic reticulum (ER) and Golgi apparatus [2]. O-glycosylation occurs in the Golgi apparatus and no lipidlinked intermediates are involved. In contrast to N-glycosylation, the process involves only the sequential addition of monosaccharides without remodeling [2,4]. Abnormalities in the synthesis of O-linked N-acetylglucosamine, galactose (Gal), mannose (Man), glucose (Glc) and fucose (Fuc) glycans have also been described [5]. GPI is a phosphoglyceride ( Figure 1) that acts as a membrane anchor of proteins to the outer leaflet of the cellular membrane. GPI-anchor biosynthesis defects (GPI-BD) are categorized by an inappropriate GPI-anchors biosynthesis or modification (protein attachment, structural remodeling at both glycan and parts of GPI). Most of the CDGs have autosomal recessive pattern of inheritance, but autosomal dominant and X-linked forms have also been described. To date, about 80 different entities have been described in correlation with congenital disorder of glycosylation or CDG-like diseases. Although some genotype-phenotype correlation has been proposed, there is a significant phenotypic variability within the same genotype.
The mechanisms of bone disease in CDGs seem to be multifactorial. Malabsorption and liver dysfunction lead to a poor nutritional state which impacts the growth hormoneinsulin-like growth factor (GH/IGF) axis and results in a short stature [9,10]. Given that patients with CDGs manifest their skeletal abnormalities in childhood, one may speculate that they are attributed to the impaired glycosylation in the bone tissue in the early stages of life. Among the different glycosylation defects, impaired protein N-glycosylation is the most common. Glycan processing (remodeling) takes place in the endoplasmic reticulum (ER) and Golgi apparatus [2]. O-glycosylation occurs in the Golgi apparatus and no lipidlinked intermediates are involved. In contrast to N-glycosylation, the process involves only the sequential addition of monosaccharides without remodeling [2,4]. Abnormalities in the synthesis of O-linked N-acetylglucosamine, galactose (Gal), mannose (Man), glucose (Glc) and fucose (Fuc) glycans have also been described [5]. GPI is a phosphoglyceride ( Figure 1) that acts as a membrane anchor of proteins to the outer leaflet of the cellular membrane. GPI-anchor biosynthesis defects (GPI-BD) are categorized by an inappropriate GPI-anchors biosynthesis or modification (protein attachment, structural remodeling at both glycan and parts of GPI). Most of the CDGs have autosomal recessive pattern of inheritance, but autosomal dominant and X-linked forms have also been described. To date, about 80 different entities have been described in correlation with congenital disorder of glycosylation or CDG-like diseases. Although some genotype-phenotype correlation has been proposed, there is a significant phenotypic variability within the same genotype.
The mechanisms of bone disease in CDGs seem to be multifactorial. Malabsorption and liver dysfunction lead to a poor nutritional state which impacts the growth hormoneinsulin-like growth factor (GH/IGF) axis and results in a short stature [9,10]. Given that patients with CDGs manifest their skeletal abnormalities in childhood, one may speculate that they are attributed to the impaired glycosylation in the bone tissue in the early stages of life.
So far, there have been no therapies targeting specific organ such as eye, kidney, heart, muscles or skeleton, with only supportive management being available [10]. This review describes skeletal manifestations and bone mineral density abnormalities in paediatric and adult CDG patients with the aim to discuss the heterogeneity of clinical manifestations and correlate the genetic data (causative genes) with observed skeletal phenotype to evaluate the potential mechanism of mutational effect on the phenotype.

Skeletal Characteristics of PMM2-CDG
PMM2-CDG is the most prevalent disorder of abnormal glycosylation of N-linked oligosaccharides leading to endocrine abnormalities including dysfunction of IGFBP3 and an acid-labile subunit in the IGF pathway which result in short stature [11]. Growth failure is commonly observed in children with PMM2-CDG [7,[12][13][14][15][16][17]. Despite normal serum calcium, phosphate and magnesium concentrations, osteopenia/osteoporosis are frequently demonstrated in this condition since childhood (Table 1) [8][9][10][11][12][13][14]. Skeletal abnormalities are common, although under-diagnosed [17], often leading to kypho/scoliosis, severe spinal cord deformity and vertebral compression fractures [7] (see Figure 2). Regular orthopaedic assessment and intervention are required if scoliosis becomes evident, with cervical spine x-rays in neutral, flexion, and extension to assess for atlantoaxial instability. Fractures are common and appear to heal normally. Skeletal dysplasia is not a typical feature of PMM2-CDG but has been reported before (Tables 1 and 2). Joint contractures are relatively common and affect patients' quality of life. cium, phosphate and magnesium concentrations, osteopenia/osteoporosis are frequently demonstrated in this condition since childhood (Table 1) [8][9][10][11][12][13][14]. Skeletal abnormalities are common, although under-diagnosed [17], often leading to kypho/scoliosis, severe spinal cord deformity and vertebral compression fractures [7] (see Figure 2). Regular orthopaedic assessment and intervention are required if scoliosis becomes evident, with cervical spine x-rays in neutral, flexion, and extension to assess for atlantoaxial instability. Fractures are common and appear to heal normally. Skeletal dysplasia is not a typical feature of PMM2-CDG but has been reported before (Tables 1 and 2). Joint contractures are relatively common and affect patients' quality of life.    Pt 1-At gestational age 26 + 3 weeks, the mother went into spontaneous labour, and the child was stillborn. Pt 2-Because of the severe kidney disease, the family decided to terminate the pregnancy at 20 + 1 weeks. Pt 3-The pregnancy was terminated at 20 weeks. [29] All-decreased ossification of the frontoparietal bones, thickening of the occipital bones, deficient ossification of cervical vertebral bodies and pubic bones, round pelvis and short tubular bones with metaphyseal flaring.
Mild phenotype [30]  severe skeletal dysplasia with radiographic pattern of Desbuquois dysplasia Pt 1-shortened limbs with hand and feet lengths <3rd pc. Spindle-shaped fingers, clinodactyly, and shortened thumbs and index. A medial deviation of the fifth toes. Wormian bones and small sella turcica, shortened long bones with large and irregular methaphyses. Pt 2-short limbs noted at 30 weeks of gestation. Several supernumerary ossification centres were noted: one next to the distal metaphysis of the humeri and another one next to the calcaneus. Autopsy showed absence of the well-defined cell columns of the proliferating and hypertrophic cartilage. The hypertrophic cell zone was reduced; the resorption zone was irregular. Primitive trabeculae were thick and misaligned.
Pt 1-died 7 days after birth of respiratory insufficiency and pulmonary hypertension.
[36] [43] Pt 2: At birth-hydrops, narrow chest, the limbs were very short in all segments with bilateral clubfeet. Radiographs showed features typical of Schneckenbecken dysplasia. The ilia were short with a snail-like appearance and a flattened acetabular roof. The long bones were short and the metaphyses were abnormally wide. The spine was abnormal, with inter-pedicular narrowing, particularly in the lumbar region.
Pt 2 died of respiratory insufficiency in the immediate neonatal period. skeletal and joint abnormalities Pt 1: 6 months-growth retardation; 1 year-remarkable joint laxity; 2 years-head circumference at 3rd pc, weight and height significantly below the 3rd pc. Her fingers were long with prominent finger pads and lax interphalangeal joints. She had an interrupted single transverse palmer crease, lax wrists, and limited elbow movements. Her toes were long with prominent pads and partial syndactyly of 3rd and 4th toes with overriding toes. Skeletal survey demonstrated bilateral proximal radioulnar synostosis and bowing of the shafts of the radius and ulna. The clavicles were short with broad medial ends. There was anterior splaying of ribs, abnormal appearance of distal metaphyses of the humeri and ulnae, cone shaped appearance of distal phalanx of first toe, and diffuse osteopenia. Pt 2: 33 years-head circumference at 3rd pc, weight at 3rd pc, height <3rd pc. Lax joints, loose skin, and limited movement of elbow joints were observed. His fingers were long and he had a single palmar crease in the left hand. He had bilateral equinovarus deformities and there were many atrophic scars on the sides of his feet and back of his legs and hips Skeletal X-rays demonstrated findings similar to those of Pt1 in particular to the proximal radioulnar synostosis bilaterally.
n.a. [6,50]   Obstetric ultrasound examination revealed macrocephaly with shortened long bones (femur length <5th pc), lumbar lordosis; Neonatal period-short long bones with, dumbbell" metaphyseal expansions, generalized epiphyseal ossification delay, ovoid and anteriorly beaked vertebral bodies, hypoplastic cervical vertebrae, hypoplastic pubic bones, and bullet-shaped short tubular bones; Cervical spine MRI: elevation of the posterior arch of C1 with the occipital bone and significant spinal canal stenosis at the craniocervical junction due to a bone spur n.a. [7] n.a. osteopenia 3 pts aged 14-27 years: Low bone density. Common clinical findings are skeletal changes including peculiar thoracic deformity and joint restriction, while a major radiological feature is diffuse osteopenia.
n.a. [17] n.a. variable skeletal anomalies 2 months-pectus excavatum, restricted extension of knees and elbows, and long fingers with camptodactyly. Diffuse osteopenia, disturbed modeling of bones: anterior widening of the ribs, thick clavicles, dorsolumbar kyphosis with slight hook-like dysplasia of the antero-inferior border of the first lumbar vertebrae. squared and flared iliac wings, horizontal acetabular roofs, and poorly formed and thickened ischial and pubic bones.
n.a. [18] n.a. variable skeletal anomalies Osteoclerosis of the femora and tibiae with narrow medullary cavities and transverse radiolucent metaphyseal lines n.a. [19] n.a. variable skeletal anomalies Bone-in-bone appearance of the skeleton, with flaring of the iliac wings and sloping acetabular roof. n.a. n.a. [52]
The features of patients with ALG9-CDG reported by Tham et al. were similar to those reported by Gillessen-Kaesbach and Nishimura and to some extent overlapping with those of individuals with ALG3-CDG and ALG12-CDG (Table 1 and 2) [27][28][29][30].
Skeletal dysplasia was also reported as the main clinical feature of patients with COG7-CDG, COG1-CDG and COG8-CDG (Table 1). While patients with COG8-CDG displayed severe skeletal dysplasia, COG1-CDG and COG8-CDG patients had a significantly milder clinical presentation (

Joint and Cartilage Involvement in CDG
In two patients with typical Schneckenbecken dysplasia (# 269250), Rautengarten et al. found biallelic mutations in both alleles of SLC35D1 (Tables 1 and 2) [43]. Schneckenbecken dysplasia (#269250) is a perinatally lethal skeletal dysplasia characterized by the distinctive, snail-like appearance of the ilia, thoracic hypoplasia, severe flattening of the vertebral bodies and short, thick long bones [44].
Brachytelephalangy was consistently found in all the reported PIGV-CDG patients ( Table 2) [52]. It was also found in most of the reported patients with PIGO-CDG (Hyper-phosphatasia with mental retardation syndrome 2, HPMRS2, #614749) [58,59]. This skeletal finding could lead to the suspicion of Coffin-Siris syndrome given the neurological findings in both syndromes, i.e., delayed psychomotor development, hypotonia and expressive language delay. Coffin-Siris syndrome in its typical form (CSS1, #135900) characterized by aplasia or hypoplasia of the distal phalanx and hypoplastic, aplastic nails (even absent fifth fingernail in some patients), developmental or cognitive delay of varying degree, distinctive facial features, hypotonia, hirsutism/hypertrichosis, and congenital malformations of the cardiac, gastrointestinal, genitourinary, and/or central nervous systems [60].

Genotype-Phenotype Correlation
The genetic background of CDG is highly variable and includes various genes. In Table 2, we report the clinical and molecular findings of CDG patients with skeletal abnormalities. Most of patients were compound heterozygotes for autosomal recessive hypomorphic mutations and homozygotes are rather sporadic or family related cases. It is difficult to draw any strong conclusions regarding the possible genotype-phenotype correlation in such a heterogenic group.
For patients with homozygous mutations, we were able to give some conclusions regarding, i.e., CSGALNACT1 and SLC35D1 genes. Knockout of the orthologous mouse CSGALNACT1 indicates that the protein is necessary for normal cartilage development and aggrecan metabolism [61]. Mutations in CSGALNACT1 are associated with a mild skeletal dysplasia and joint laxity [41]. The clinical significance of the described variant c.791A > G (p.Asn264Ser) is defined as likely pathogenic.
Hiraoka et al. created Slc35d1-deficient mice that present with a lethal form of skeletal dysplasia (severe shortening of limbs and facial structures) with short, sparse chondroitin sulphate chains caused by a defect in chondroitin sulphate biosynthesis [62]. Epiphyseal cartilage in these homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. Variant c.125delA in SLC35D1 was reported only once, and its significance has been defined as pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/RCV000001182, accessed on 27 June 2021).
Skeletal and/or bone mineral density abnormalities are common features of all CDG types. Osteoporosis and/or osteopenia, growth failure, skeletal dysplasia and joint abnormalities were described (Tables 1 and 2). Genotype-phenotype correlation is impossible due to gene heterogeneity, diversity of individual markers and compound heterozygosity in most described cases (Table 2).
Glycosylation is essential for proteins involved in the development of cartilage and bone and in skeletal patterning pathways. Two of the glycosylation defects, TMEM165-CDG and CSGALNACT1-CDG, are characterized by skeletal dysplasia and abnormal cartilage development. It has been also observed in an experimental study by Bammens et al. (2015), that the TMEM165-deficient zebrafish model exhibited phenotypic patterns of bone dysplasia and abnormal cartilage development similar to the major clinical findings reported in three patients with a homozygous splice mutation [63].
Two other CDGs, CSGALNACT1-CDG and SLC35D1-CDG, are caused by defects in genes responsible for glycosaminoglycans biosynthesis.
CSGalNAcT1 (chondroitin sulphate N-acetylgalactosaminyltransferase-1) gene codes the CSGalNAcT-1 protein that is the main enzyme in the biosynthesis of sulphated glycosaminoglycans chondroitin and dermatan sulphate in cartilage [42]. Biallelic loss-offunction mutations in CSGALNACT1 gene results in reduced CSGalNAcT-1 activity leading to altered levels of chondroitin, dermatan, and heparan sulphate and cause a mild skeletal dysplasia with joint laxity and advanced bone age, CSGALNACT1-CDG [42].
Another gene, SLC35D1 gene, encodes the protein that transports substrates needed for glucuronidation and chondroitin sulphate biosynthesis [62]. The protein resides in the endoplasmic reticulum, and transports both UDP-glucuronic acid and UDP-Nacetylgalactosamine from the cytoplasm to the endoplasmic reticulum. In humans, loss of function mutation in this gene cause Schneckenbecken dysplasia associated with perinatal lethal skeletal dysplasias [62].

Risk Factors for Osteoporosis in CDGs
Hormonal dysfunction, resulting from hypogonadotrophic hypogonadism, is a risk factor for reduced bone mineral density in females with CDGs [7,64,65]. It requires treatment with oestrogen to avoid osteoporosis; however, it increases the risk of thrombotic events and must be dosed carefully.
Gastrointestinal problems and malabsorption may lead to hypovitaminosis D. It is therefore worth considering screening for common causes of chronically decreased vitamin D such as celiac disease [65].
Bone deformities and chronic pain result in limited mobility and wheelchair use. Low BMI and disuse sarcopenia are frequent clinical features of patients with CDGs and, in the long term, they may cause low bone mineral density and recurrent fractures.

Bone Markers
Historically, this GPI-linked enzyme, has been used to assign glycosylphosphatidylinositol biosynthesis defects (GPI-BD) to the phenotypic group termed hyperphosphatasia with mental retardation syndrome (HPMRS) and to distinguish them from another subset of GPI-BD-multiple congenital anomalies hypotonia seizures syndrome (MC-AHS) [51]. However, with the increasing number of individuals diagnosed with a GPI-BD, hyperphosphatasia has been shown to be a variable feature and not more useful for GPI-BD distinguishing.
Bone-specific alkaline phosphatase activity measured in serum is an indicator of osteoblastic activity.
Serum and urinary deoxypyridinoline and pyridinoline, cross-linking amino acids that stabilize collagen chains within the extracellular matrix, can also be measured when screening for reduced bone mineral density in PMM2-CDG disorders [10]. Bone markers of formation (P1NP) or resorption (CTX) measurement in CDGs requires further clinical studies to better understand what factors are likely to affect their concentration in these conditions. All the bone markers should be interpreted in the context of symptoms, age, gender, limited physical activity including wheelchair use, and diet.
Transferrin glycosylation has been the main biomarker for CDG screening and monitoring of the response to treatment. The analysis of protein N-glycosylation profile is widely used in CDGs and other conditions [66].
There is a scope for using N-glycans in monitoring of bone disease progression and further research is warranted to confirm the preliminary findings [67].
The effectiveness of supportive management can be evaluated by DEXA scan (Figure 3), a gold standard method for monitoring the progression of bone mineral density decline. The whole spine x-ray ( Figure 2) can be used for identification of bone deformities and fractures. QCT however is the sensitive quantitative method used to assess the bone mineral density. Body composition has become increasingly utilized in Inherited Metabolic Disorders field and provides additional information about the bone and muscle involvement.
The effectiveness of supportive management can be evaluated by DEXA scan (Figure  3), a gold standard method for monitoring the progression of bone mineral density decline. The whole spine x-ray ( Figure 2) can be used for identification of bone deformities and fractures. QCT however is the sensitive quantitative method used to assess the bone mineral density. Body composition has become increasingly utilized in Inherited Metabolic Disorders field and provides additional information about the bone and muscle involvement.
Importantly, a multidisciplinary approach with pain specialist, rheumatologist, endocrinologist, metabolic specialist, physiotherapist and dietician is key to successful management of bone health in this group of disorders.

Therapies and Bone Health
Some CDG types including PGM1-CDG and TMEM165-CDG, characterized by an undergalactosylation of the N-glycans have shown good responses to galactose supplementation [68,69]. Additionally, deficient import of UDP-Gal into the ER or the Golgi, required by β1,4-galactosyltransferase to elongate the growing glycan chain, has been documented in SLC35A2-CDG [70]. Although in this condition, galactose supplementation has improved glycosylation, it has not been clear whether it has had an impact on clinical manifestations of the disease including bones anomalies [70]. Apart from galactose, mannose has been utilized as a therapy in MPI-CDG [9,10] and has been shown to Importantly, a multidisciplinary approach with pain specialist, rheumatologist, endocrinologist, metabolic specialist, physiotherapist and dietician is key to successful management of bone health in this group of disorders.

Therapies and Bone Health
Some CDG types including PGM1-CDG and TMEM165-CDG, characterized by an undergalactosylation of the N-glycans have shown good responses to galactose supplementation [68,69]. Additionally, deficient import of UDP-Gal into the ER or the Golgi, required by β1,4-galactosyltransferase to elongate the growing glycan chain, has been documented in SLC35A2-CDG [70]. Although in this condition, galactose supplementation has improved glycosylation, it has not been clear whether it has had an impact on clinical manifestations of the disease including bones anomalies [70]. Apart from galactose, mannose has been utilized as a therapy in MPI-CDG [9,10] and has been shown to restore endocrine function and coagulation and alleviates enteropathy, but does not always reverse progressive hepatic involvement [71]. The long-term effect of mannose on skeletal outcomes needs careful attention. As a result of the UDP-fucose transporter defect and impaired fucosylation in the Golgi in SLC35C1-CDG, patients affected with this CDG type have been benefited from fucose supplementation [72,73]. A decreased infection rates and improved expression of E-and P-selectin ligands and restored neutrophil numbers have been documented [72,73].
Although biochemical and haematological parameters improved with these therapeutic approaches, the impact on bone tissue remains unclear. Long-term prospective observational studies will elucidate whether the bone health in CDGs improves with the nutritional supplementation and/or new therapeutic approaches including pharmacological chaperone, antisense or gene therapies (discussed in detail in [9,10]).

Conclusions
CDGs should be considered as a differential diagnosis of skeletal dysplasia, especially in multiorgan and systemic involvement. It is particularly important for CDGs with prenatal-onset skeletal dysplasia which is associated with severe clinical outcomes. Thus, early diagnosis is key to family genetic counselling.
Skeletal involvement in CDGs is underestimated and thus should be more carefully investigated and managed shortly after a diagnosis is made. In adult CDG patients screening for additional risk factors for osteoporosis should be considered. There is a scope for utilizing bone markers in monitoring of the bone tissue dynamics. The anticipated expansion in the therapeutic strategies is hoped to impact the bone health in CDGs and prevent skeletal complications.  Data Availability Statement: All data generated or analysed during this study are included in this published article.

Conflicts of Interest:
The authors declare no conflict of interest.