In addition two genes on X chromosome are identified as etiologies of OI; increasing the number of genes for OI to 18. Loss-of-function mutations in PLS3 encoding plastin-3 were discovered as a cause of one form of X-linked osteoporosis with fractures (van Dijk et al., 2013). Recently, Lindert et al., 2016 identified an X-linked form of osteogenesis imperfecta in two independent pedigrees. Phenotypic inheritance pattern, linkage analysis and next generation sequencing (NGS) were used to localize the causative gene in each family to MBTPS2 at Xp22.

The first 4 subtypes of OI (clinical phenotypes) are caused by COL1A1 and COL1A2 genes. Further numbering upto XVII types (showing clinical overlap with initial four subtypes) is included in OMIM based on the causative genes and all of them are autosomal recessive inheritance except OI V. Data from India shows that COL1A1 and COL1A2 mutations contribute to only 70% of cases with OI (Stephen et al., 2014). High level of consanguinity is probably the main contribute to the higher prevalence of autosomal recessive OI. A small number of cases with recessive OI are reported from India (Stephen et al., 2015). The products of the genes for recessive types of OI are mostly enzymes which involves in the post translational modification of the pre pro type I collagen produced from COL1A1 and COL1A2 genes. The functions of these genes include lysyl 3 hydroxylation, collagen processing and maturation, collagen stability and bone formation and homeostasis. More genes are still getting identified. These disorders being very rare most of the publications report only a few cases. There is no large scale case series involving study of multiple genes.

As can be seen in table 1, the functions of the genes causing OI are collagen synthesis, modification, folding and crosslinking. In addition to mutations in COL1A1 and COL1A2 genes, defects in osteoblast development like CREB3L1 and WNT1 also cause OI. CRTAP, LEPRE1 and PPIB are important proteins of the complex involved in hydroxylation of propyl 3 complex which plays a critical role for the proper collagen helix formation in the cell. Improved understanding of functional pathways of collagen is paving ways to different treatment strategies.

5 Approach to a Case of OI

Clinical diagnosis of a most of the cases is relatively easy and straightforward. Radiological evidence of decreased bone density along with history of repeated fractures and / or fractures with trivial trauma suggests the diagnosis. Presence or absence of blue sclera, dentiginous imperfecta, large open fontanelles, wormian bones and pre-senile deafness helps in the diagnosis and clinical classification. Other causes of decreased bone density need to be ruled out by associated findings and investigations (Table 2).

Differential diagnoses for antenatal cases: In utero, short and undermineralised bones, fractures can be appreciated in utero and lead to suspicion of OI. The following conditions need to be ruled alongwith–

⋅ Hypophasphatasia - characterized by defective mineralization of bone and/or teeth in the presence of low activity of serum and bone alkaline phosphatase. Clinical features range from stillbirth without mineralized bone at the severe end to pathologic fractures of the lower extremities in later adulthood at the mild end.

⋅ Thanatophoric dysplasia- neonatal lethal short-limbed dwarfing condition, well-ossified spine and skull, platyspondyly, ventriculomegaly, narrow chest cavity with short ribs, polyhydramnios, and bowed femurs (TD type I), cloverleaf skull (kleeblattschaedel) (often in TD type II; occasionally in TD type I) and/or relative macrocephaly.

⋅ Campomelic dysplasia- skeletal dysplasia characterized by distinctive facies, Pierre Robin sequence with cleft palate, shortening and bowing of long bones, and club feet. Other findings include laryngotracheomalacia with respiratory compromise and ambiguous genitalia or normal female external genitalia in most individuals with a 46,XY karyotype.

⋅ Achondrogenesis - extremely short limbs with short fingers and toes, hypoplasia of the thorax, protuberant abdomen, and hydropic fetal appearance caused by the abundance of soft tissue relative to the short skeleton.

Family history is important in the evaluation of a case with suspected OI, as milder/mosaic/asymptomatic forms are difficult to detect clinically. A three generation pedigree should be drawn and the family should be asked for history of recurrent easy fractures, short stature, presenile deafness in any of the family members. Presence of consanguinity suggests the possibility of an autosomal recessive disorder. In some cases the definitive diagnosis of OI may be difficult and confirmation by mutation testing is essential.

6 Molecular Diagnosis

Mutation detection confirms the diagnosis and is essential for genetic counseling regarding risk of recurrence and preventing recurrence by way of prenatal diagnosis. The heterogenic etiology and large sizes of genes makes mutation detection a complex process. The causative gene mostly cannot be predicted based on the clinical features as there is no genotype phenotype correlation. Presence of hypertrophic callus and ossification of interosseous membrane is characteristic of OI type V for which only one causative mutation (c.-14C>T in IFITM5 gene) is reported in all the cases. The severity of the presentation also does not provide any clue the causative gene. Initially when COL1A1 and COL1A2 were the only known genes, collagen analysis was done to decide the possible causative gene. Later, DHPLC (Denaturing High Performance Liquid Chromatography) was used as a screening technique to identify the location of mutation and then the specific exons were sequenced. Such type of screening was essential as both the genes are large genes with 51 and 52 exons in COL1A1 and COL1A2 respectively. Identification of newer genes for OI has increased the complexities of molecular diagnosis. Next Generation Sequencing (NGS) is of great help in this disorder associated with a large number of genes, some of which have a large number of exons. A panel of all OI genes or sequencing of all genes of clinical relevance (Exome sequencing) is the test of choice. The studies on the detection rates and relative contribution of various genes to OI are still not available. Published data about mutations in genes other than COL1A1 and COL1A2 is limited worldwide and hence, more information about phenotypes of recessive OI is needed.

7 Management

The basic serum chemistries- serum calcium, phosphate, vitamin D and alkaline phosphatase are normal in patients with OI. However these values might be slightly elevated after a fracture. But as the patients started on bisphonates, grow older, have sedentary lifestyle due to bony deformities and fractures, the calcium and vitamin D levels fall. This can lead to delayed healing of fractures and increased bone fragility. So, it is recommended to supplement calcium and vitamin D in patients with OI according to the age group. There is still no “optimal treatment” for OI, both for limiting or preventing fractures and pain relief and improved mobility. Also, critical assessment of treatment outcomes is limited by the small numbers of participants in clinical trials and the short duration of many trials, which is frequently limited to 1 or 2 years of observation. The following table provides the list of various treatment modalities (Table 3).

7.1 Existing treatment strategies

1. Bisphosphonates- Bisphosphonates, oral and intravenous decrease osteoclastic bone resoprtion. Various studies have shown variable efficacy of bisphosphonates on decreasing the fracture frequency and symptomatic relief of pain. The variability of response may be due to differential effects of bisphosphonates on various types of OI; the issue which may be solved with studies on mutation proved cases of OI. For example, we found that bisphosphonates increased callus formation in a case with type V OI (Dwan et al., 2014).

The findings of the 2014 Cochrane review (Dwan et al., 2014) for oral and intravenous Bisphosphnates in OI can be summed up as follows-

a. The oral or intravenous bisphosphonates increase bone mineral density in children and adults with OI, the effect not being different with the different bisphosphonates.

b. It is unclear whether oral or intravenous bisphosphonate treatment consistently decreases fractures, though there is no increased fracture rate.

c. The studies included in the Cochrane review do not show bisphosphonates conclusively improve clinical status (reduce pain; improve growth and functional mobility) in people with OI.

2. Teriparatide- Teriparatide is human recombinant parathyroid hormone, which increases bone mass by increasing osteoblast bone formation. It is highly effective in the treatment of age-related osteoporosis.

The few randomized trials of teriparatide in OI patients show increased BMD, especially in Type 1 OI than in individuals with OI types IV and III, but there was no significant decrease in number of fractures (Orwoll et al., 2014).

3. Denosumab (anti-RANK-ligand antibody)- The RANK, RANKL complex regulates bone-remodeling cycles by regulating osteoblast/osteoclast coupling and osteoclast differentiation. RANK is present on the osteoclast precursor, and RANKL produced by the osteoblast is part of the TNF superfamily and, along with the soluble decoy receptor osteoprotegerin, are essential regulators of osteoclast development and function. Denosumab is a human monoclonal antibody to RANKL; studies involving age-related osteoporosis have showed the efficacy of denosumab in reducing signaling via RANK, leading clinically to prevention of bone loss (Hoyer-Kuhn et al., 2014).

Due to poor response to Bisphsphonates in OI Type VI, Denosumab was tried with success in OI Type VI leading to BMD increase, normalization of vertebral shape, and decrease in fracture rate. Denosumab treatment also improved BMD and longitudinal bone growth in two children with COL1A1/A2 mutations previously treated with Bisphosphonates.

7.2 New therapeutic approaches

1. Antisclerostin antibody - Sclerostin is a negative regulator of bone formation released from osteocytes that modulates osteoblast activity acting through Wnt/β-catenin pathway. Preclinical studies have demonstrated that treatment with antisclerostin monoclonal antibody acts as osteoanabolic therapy improves bone mass and bone strength, and enhances repair of fractures in animal models.

2. Cathepsin K antibody - Cathepsin K is highly expressed in osteoclasts and is an essential enzyme involved in the degradation of type I collagen in the organic bone matrix. In an animal model, the cathepsin K monoclonal antibody (Odanacatib) effectively suppressed bone resorption. A phase III randomized, placebo-controlled trial assessed the effect of Odanacatib on fracture risk over 5 years of treatment in women with osteoporosis, has shown increase in BMD and a significant reduction in the risk of fractures. Applicability to the collagen defect in OI remains to be determined.

3. Transforming growth factor-β - TGF-β is produced by osteoblasts and acts to coordinate bone remodelling by coupling osteoblasts and osteoclasts in the process of bone remodelling. TGF-β is secreted predominantly in an inactive latent form and is deposited into the bone matrix. It has been reported that excessive TGF-β signaling is a mechanism of OI in both recessive and dominant OI mouse models. Also, treatment of mice with the anti-TGF-β neutralizing antibody 1D11 corrected the bone phenotype and improved lung abnormalities in both recessive and dominant forms of OI.

4. Prenatal and postnatal transplantation of mesenchymal stem cells - Severe to lethal forms of OI may be diagnosed in utero by ultrasonography starting at the 16th week. Following prenatal and postnatal cell transplantation in OI improvement was seen in linear growth and fractures were reduced in number, in fetus, neonatal and later life. In humans, improvement of linear growth and reduction of fracture rate followed prenatal and postnatal cell transplantation in OI. Additionally, prenatal transplantation of allogeneic MSCs in three OI pregnancies indicated that it has appeared to be safe. A clinical trial in human pregnancy is currently in progress. (Westgren et al., 2015).

7.3 Multidisciplinary management

Orthopedic management might be necessary in cases of severe bone deformity impairing function, with recurrent fractures and nonunion of fractures. To date, there are no physiotherapeutic treatment protocols available for children and adults with OI. A recent study investigated a rehabilitation approach combining resistance training, body-weight-supported treadmill training, and neurodevelopmental treatment associated with side-alternating whole-body vibration in 53 individuals with OI (ages 2.5–24.8 years) for 6 months within a period of 12 months of treatment. There was improvement of mobility between, and also an increase in lean mass and BMD was observed (Hoyer-Kuhn et al., 2014). Further studies are needed to address the role of rehabilitation in OI patients.

8 Genetic Counseling and Prenatal Diagnosis

Bisphosphonates has given some relief to some patients of OI; though for many cases the life continues to be painful and handicapping. Molecular diagnosis can differentiate between OI inherited in dominant or recessive fashion and accurate risk of recurrence can be provided to the families. Mutation based prenatal diagnosis can be provided at early gestation and to all families. In situations without molecular diagnosis, ultrasonographic based diagnosis before 20 weeks of gestation for case with lethal variety of OI. For other varieties, shortening and bending of femora may be seen in some cases and may be in the later part of pregnancy. However, normal length and shape of long bones in a fetus cannot rule out OI.

9 Conclusion

Last decade has improved understanding of genetics of OI due to identification of many more genes for OI. NGS based diagnostics has provided simple strategy in clinical settings and is also identifying new genetic etiologies in research settings. Autosomal recessive OI is probably more common in India due to high prevalence of consanguinity and makes molecular diagnosis of each case essential as the risk of recurrence is 25% and phenotype is usually severe in recessive varieties of OI. New treatments may provide specific drug for specific type of OI and improve the outcome.

References