Osteogenesis imperfecta (OI) comprises a heterogeneous group of diseases characterized by susceptibility to bone fractures with variable severity and, in most cases, with presumed or proven defects in collagen type I biosynthesis (Van Dijk et al., 2010). Other clinical manifestations include short stature, blue sclerae, dentinogenesis imperfecta, joint laxity and variable degree of postpubertal hearing loss and presence of wormian bones on skull radiographs (Martin and Shapiro, 2007; Van Dijk et al., 2011). Bone pain is a frequent symptom (Seikaly et al., 2005).
OI is inherited in both autosomal dominant and recessive inheritance patterns. The large majority of OI cases follow a dominant pattern of inheritance or are sporadic and result from heterozygous mutations in COL1A1 (OMIM 120150) or COL1A2 (OMIM 120160), the genes that encode the proαl(I) and proα2(I) chains of type I collagen, the major structural protein of bone (Ben Amor et al., 2011). The pro-alpha chains of type I collagen contain an uninterrupted helical region consisting of 338 repeats of the Gly-Xaa-Yaa triplet. Biosynthesis of procollagen is a complex process that requires several cotranslational and post-translational modifications that stabilize collagen helical folding (Myllyharju and Kivirikko, 2004; Cabral et al., 2014). Glycine substitutions in the collagen alpha chains delay folding and increase exposure to modifying enzymes, resulting in collagen overmodification. Some OI cases (≈10%) have recessive inheritance, caused by deficiency of proteins that interact with collagen for folding or post-translational modification (Forlino et al., 2011).
More recently, another gene, IFITM5, which is thought to be involved in osteoblast maturation, has also been associated with dominant OI after the identification of a recurrent mutation in this transcript in a small number of families (Cho et al., 2012; Semler et al., 2012). In contrast, recessively inherited OI results in many cases from homozygous or compound heterozygous mutations in a rapidly growing list of genes participating in post-translational processing and modification of type I collagen, the formation of procollagen, and osteoblast differentiation. These recessive genes are CRTAP (Morello et al., 2006), LEPRE1 (Cabral et al., 2007), PPIB (Barnes et al., 2010), OSX (Lapunzina et al., 2010), BMP1 (Asharani et al., 2012; Martinez Glez et al., 2012), SERPINH1, SERPINF1, TMEM38B (Shaheen et al., 2012), FKBP10 (Barnes et al., 2012), the telopeptide lysyl hydroxylase PLOD2 (Puig Hervás et al., 2012), PLS3, CREB3L1 and WNT1 (Fahiminiya et al., 2013; Keupp et al., 2013).
Several classification systems have been proposed for OI, based on clinical, radiological and, recently, molecular findings. The most commonly used classification was proposed by Sillence et al. (1979). It includes four types based on clinical criteria: types I–IV. OI type I is the mildest form. OI type II is usually severe and lethal during the perinatal period. OI type III is the most severe form of OI that is compatible with life, and OI type IV is a moderately severe form of OI with multiple fractures and mild to moderate bone deformities. Sclerae in this type are white or grey. Aglan et al. (2012) provided a more quantitative clinical scoring system (CSS) to assess the severity and to help in follow-up of OI patients. The proposed scoring system included five major criteria of high prognostic value: number of fractures per year, motor milestones, bone deformities, height and bone density. Each criterion was assigned a score from 1 to 4 and each patient was put on a scale from 1 to 20 according to these five criteria.
Here we report on the phenotypic and mutational spectrum of 11 Egyptian patients with autosomal dominant osteogenesis imperfect (AD-OI) carrying pathogenic mutations in the COL1A1 gene. This study is the first to report the mutational spectrum of AD-OI among Egyptians.
Patients and methods
The study included 11 patients from four unrelated Egyptian families with AD-OI referred to the Limb Malformations and Skeletal Dysplasia Clinic (LMSDC), Centre of Medical Excellence, National Research Centre. All patients were subjected to meticulous clinical evaluation including history, pedigree analysis and detailed physical examination and anthropometric measurements. Investigations including skeletal radiological survey and bone densitometry were performed for all patients. OI classification was done according to Sillence et al. (1979). The degree of severity was assessed according to the criteria reported by Aglan et al. (2012). When the diagnosis of OI was confirmed, the probable mode of inheritance had been determined. Patients with suspected AD-OI were subjected to molecular studies.
Mutation analysis of COL1A1 and COL1A2 genes
Genomic DNA was extracted from peripheral blood lymphocytes of patients and their parents after obtaining signed informed consent according to the guidelines of the Medical Ethics Committee of the National Research Centre (NRC). The entire coding regions of COL1A1 and COL1A2 genes were amplified using multiple overlapping primers designed by Primer 3 Input Software, version 0.4.0. The coding regions and their exon/intron boundaries of ∼50 bp sequence were investigated to identify any splice site variation as well. The sequence of primers is available upon request from the corresponding author. Our standard PCR cycling conditions were as follows: initial denaturation at 96°C for 5 min; 30 cycles of denaturation at 96°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 30 min; and an additional extension at 72°C for 5 min. The PCR products were purified by a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and directly sequenced in both directions using the Big Dye Termination kit (Applied Biosystems, Foster City, California, USA) and analysed on the ABI Prism 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s instructions. Mutations were named according to HGVS guidelines using NM_000088.3 as the reference sequence. The A of the first ATG was considered as nucleotide +1.
We investigated 11 patients from four unrelated families with AD-OI. Parental consanguinity was negative in all studied probands. Pedigrees of the studied families are shown in Fig. 1. Diagnosis of OI was confirmed by the presence of blue sclera, recurrent fractures and characteristic radiological signs in the form of bony fractures, callus formation and skull wormian bones (Fig. 2), in addition to low bone density assessed by bone densitometry. Patients were classified according to Sillence into four families with OI type I and one family with OI type III. All patients had a CSS score ranging from 5 to 9, except for one patient (type III) who showed a high score of 16–17 (Table 1).
Mutation analysis of all coding exons of COL1A1 and COL1A2 revealed four distinct pathogenic mutations in COL1A1 (Fig. 3 and Table 1). In contrast, no mutations were identified in COL1A2. The identified COL1A1 mutations were distributed all over the gene and included one missense (p.G26D) and three nonsense (p.Q822*, p.R1026* and p.R882*) mutations. All mutations segregated with the disease in all four families; that is, the mutation was found in one of the affected parents, whereas the other was normal at the site of the mutation.
OI is a genetic disorder characterized by bone fragility and fractures. The clinical features range from mild symptoms to severe bone deformities and neonatal lethality. OI is inherited in both recessive and dominant patterns. The majority of OI cases follow a dominant inheritance and result from mutations in COL1A1 or COL1A2. To date, more than 1000 distinct variants in the COL1A1 and COL1A2 genes have been identified that give rise to OI (http://oi.gene.le.ac.uk). In contrast, recessively inherited OI results in many cases from homozygous or compound heterozygous mutations in a rapidly growing list of genes participating in post-translational processing and modification of type I collagen, the formation of procollagen, and osteoblast differentiation. These recessive genes are CRTAP (Morello et al., 2006), LEPRE1 (Cabral et al., 2007), PPIB (Barnes et al., 2010), OSX (Lapunzina et al., 2010), BMP1 (Asharani et al., 2012; Martinez Glez et al., 2012), TMEM38B (Shaheen et al., 2012), SERPINH1, SERPINF1, PLS3, CREB3L1, FKBP10 (Barnes et al., 2012), the telopeptide lysyl hydroxylase PLOD2 (Puig Hervás et al., 2012) and WNT1 (Fahiminiya et al., 2013; Keupp et al., 2013). From our experience in the LMSD clinic we observed that the frequency of autosomal recessive-inherited OI in our population was 35% of the referred OI patients (unpublished data) in contrast to 5–10% in western countries. This can be explained by the high consanguinity rate in our population (Temtamy and Aglan, 2012).
According to Sillence et al. (1979) OI is classified into four types (I, II, III and IV) (OMIMs: 166200, 166210, 259420 and 166220) on the basis of clinical presentation, radiographic findings and severity. This classification has been expanded to include, at present, 17 genetic causes of OI and closely related disorders (Van Dijk and Sillence, 2014). However, in the latest nosology and classification of genetic skeletal disorders, Warman et al. (2011) revealed the genetic complexity of the molecular bases of OI with clear documentation of the extensive phenotypic variation arising from a single locus. The authors agreed upon retaining the Sillence classification as the prototypic and universally accepted way of classifying the degree of severity. More recently, Van Dijk and Sillence (2014) focused on the clinical classification of OI and returned to a descriptive and numerical grouping of five OI syndromic groups. Given the high heterogeneity in this group of patients, Aglan et al. (2012) provided a quantitative CSS and recommended its use in complement with the Sillence classification and molecular studies.
When the Sillence classification was applied to the studied patients, OI-I was found to be the most common among the referred patients (nine out of the 11 patients with mild CSS ranging from 5 to 9), followed by OI-III, which was found in two patients with severe CSS 16–17 due to more fractures, bony deformities secondary to these fractures, scoliosis, short stature and reduced bone density. Height was decreased with the increased number of fractures. There was a parallel relation between the severity and the degree of short stature. Bone mineral density was significantly decreased in patients with OI-III compared with patients with OI-I in which there was osteopenia rather than osteoporosis. Our patient with OI-III had severe pain, whereas patients with OI-I experienced minimal pain mainly in the back. The more severe the pain, the more reduced the Dual-energy X-ray absorptiometry. Blue sclera was evident in all of our affected patients. The application of CSS to the studied patients provided an easy, simple, quantitative tool for assessment of disease severity as stated by Aglan et al. (2012).
Mutational analyses of the COL1A1 and COL1A2 genes in the studied OI patients revealed four different pathogenic COL1A1 mutations distributed across the gene. The mutations identified were three nonsense mutations (Q822*, R882* and R1026*) and one missense (G26D) mutation. Q822*, R882* and R1026* are protein truncating mutations and are predicted to result in nonsense-mediated mRNA decay. They are associated with a milder phenotype and a CSS ranging from 5 to 9 (Table 1). This is in accordance with previous studies reporting that milder forms of OI generally result from mutations that lead to the loss of mRNA from one COL1A1 gene (Ben Amor et al., 2011). The Q822* mutation is an extremely rare mutation and was reported before in one Chinese patient with OI type I (Liu et al., 2007). In contrast, the R1026* mutation found in the twin patients of family III is a common COL1A1 mutation. It has been reported before in nine patients of various ethnic groups like Syria, Israel, Finland, China and Italy (Ries et al., 2000; Hartikka et al., 2004; Ries Levavi et al., 2004; Fuccio et al., 2011; Valentina Gentile et al., 2012; Zhang et al., 2012). Interestingly, it is predominantly associated with OI type I.
Regarding other genotype/phenotype correlations, the proband of family I with OI type III was severely affected with a CSS of 16–17. This family carried a previously reported missense mutation in the first exon of the gene that involves the substitution of glycine by aspartic acid. Mutations resulting in substitution of glycine residues in almost any of the 338 Gly-X-Y tripeptide motifs of the gene are usually associated with severe forms of OI, including bone deformity, increased fracture rate and short stature (Marini et al., 2007; Bodian et al., 2009 as seen in our patient). Interestingly, the G26D mutation found in family I was described before in two patients listed in the COL1A1-Osteogenesis Imperfecta Variant Database (http://oi.gene.le.ac.uk). One of these two patients had OI type I, whereas the second patient had OI type III (Fuccio et al., 2011), thus with no consistent genotype/phenotype correlation.
OI is a complex hereditary disease with a remarkable clinical variability warranting a logical classification system. Application of the CSS scoring system is recommended in complement with the Sillence classification and molecular studies for proper genetic counselling. Report of the phenotypic and mutational spectrum of 11 patients with AD-OI identified four distinct mutations in the COL1A1 gene with no common or hotspot mutation, and, although the number of studied patients is low, it reflects a genetic heterogeneity of the disease in Egyptian patients. Investigation of a larger number of patients with AD-OI is warranted to shed light on the mutation pattern of COL1A1 and COL1A2 and throw more light on genotype–phenotype correlations. Knowledge of the specific molecular genetic cause is the starting point for the development and assessment of therapy in patients with heritable disorders including OI. This study is the first to report the mutational spectrum of autosomal dominant OI among Egyptians.
This work was funded by a research grant from NRC (Project Number: 10010603). The authors are grateful to all patients and their families for participating in this study.
Conflicts of interest
There are no conflicts of interest.
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