Targeted sequencing of a pediatric metabolic bone gene panel using a desktop semiconductor next-generation sequencer.

Metabolic bone disorders in children frequently are heritable, but the expanding number of genes associated with these conditions makes it difficult to perform molecular diagnosis. In the present study, we therefore evaluated a semiconductor (SC)-based sequencing system for this purpose. A total of 65 DNA samples were analyzed comprising 24 samples from patients with 27 known pathogenic mutations, 6 samples from patients with prior negative Sanger sequencing, and 35 consecutive samples from patients with suspected heritable metabolic bone disorders who had not had prior molecular diagnosis. In the samples with known pathogenic mutations, 26 of 27 mutations were identified by SC sequencing. All single nucleotide variants were correctly identified, but a 7-nucleotide duplication in CYP27B1 was not detected. SC sequencing revealed two pathogenic mutations in the six samples where prior Sanger sequencing had failed to identify a mutation. Finally, pathogenic mutations were found in 27 samples of patients with unknown mutation status (15 in COL1A1, 9 in COL1A2, 1 in LEPRE1, 1 in LRP5, 1 in PHEX). Subsequent Sanger sequencing confirmed the mutations in all 27 samples. In conclusion, we found that SC sequencing is suitable for the diagnosis of heritable metabolic bone disorders in children.

Osteogenesis imperfecta type V: marked phenotypic variability despite the presence of the IFITM5 c.-14C>T mutation in all patients.

BACKGROUND: Osteogenesis imperfecta (OI) type V is an autosomal dominant bone fragility disorder that we had described a decade ago. Recent research has shown that OI type V is caused by a recurrent c.-14C>T mutation in IFITM5. In the present study, we assessed all patients diagnosed with OI type V at our institutions for the presence of the IFITM5 mutation. METHODS: IFITM5 exon 1 was analysed by Sanger sequencing in genomic DNA from 42 patients with OI type V (age: 2-67 years; 18 female). RESULTS: The c.-14C>T mutation of IFITM5 was detected in all individuals. Indicators of disease severity varied widely: Height z-scores (n=38) ranged from -8.7 to -0.1, median -3.5. Median final height was 147 cm in men (N=15) and 145 cm in women (N=10). Lumbar spine areal bone mineral density z-scores in the absence of bisphosphonate treatment (n=29) were between -7.7 and -0.7, median -5.3. Scoliosis was present in 57%, vertebral compression fractures in 90% of patients. CONCLUSIONS: Even though the disease-causing mutation is identical among patients with OI type V, the interindividual phenotypic variability is considerable.

Genotype-phenotype correlations in nonlethal osteogenesis imperfecta caused by mutations in the helical domain of collagen type I.

Osteogenesis imperfecta (OI) is a heritable disorder with bone fragility that is often associated with short stature, tooth abnormalities (dentinogenesis imperfecta), and blue sclera. The most common mutations associated with OI result from the substitution for glycine by another amino acid in the triple helical domain of either the alpha1 or the alpha2 chain of collagen type I. In this study, we compared the results of genotype analysis and clinical examination in 161 OI patients (median age: 13 years) who had glycine mutations in the triple helical domain of alpha1(I) (n=67) or alpha2(I) (n=94). Serine substitutions were the most frequently encountered type of mutation in both chains. Compared with patients with serine substitutions in alpha2(I) (n=40), patients with serine substitutions in alpha1(I) (n=42) on average were shorter (median height z-score -6.0 vs -3.4; P=0.005), indicating that alpha1(I) mutations cause a more severe phenotype. Height correlated with the location of the mutation in the alpha2(I) chain but not in the alpha1(I) chain. Patients with mutations affecting the first 120 amino acids at the amino-terminal end of the collagen type I triple helix had blue sclera but did not have dentinogenesis imperfecta. Among patients from different families sharing the same mutation, about 90 and 75% were concordant for dentinogenesis imperfecta and blue sclera, respectively. These data should be useful to predict disease phenotype in newly diagnosed OI patients.

Relationship between genotype and skeletal phenotype in children and adolescents with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that in the majority of cases is caused by mutations in COL1A1 or COL1A2, the genes that encode the two collagen type I alpha chains, alpha1(I) and alpha2(I). In this study, we examined the relationship between collagen type I mutations and bone densitometric and histomorphometric findings in pediatric OI patients who had not received bisphosphonate treatment. Lumbar spine areal bone mineral density (LS aBMD) was measured in 192 patients (99 girls, 93 boys; age range 3 weeks to 16.9 years) who had either COL1A1 mutations leading to haploinsufficiency (n = 52) or mutations that lead to the substitution of glycine by another amino acid in the triple-helical domain of either the alpha1(I) (n = 58) or the alpha2(I) chain (n = 82). Compared with patients with helical mutations, patients with COL1A1 haploinsufficiency on average were taller and heavier and had higher LS aBMD. After adjustment for age, sex, and height Z-scores, the mean LS aBMD Z-scores were -4.0 for the haploinsufficiency group and -4.7 for both helical mutation groups. In the whole patient population, the average LS aBMD Z-score was higher by 0.6 (95% confidence interval 0.2-1.0) in girls than in boys. Iliac bone histomorphometry (in a subgroup of 96 patients) showed that outer bone size (core width) and trabecular bone volume were similar between genotypic groups, but cortical width was 49% higher in the haploinsufficiency group compared with patients with helical mutations in alpha2(I). Bone turnover parameters were lower in the haploinsufficiency group than in patients with helical mutations. In the group of patients with helical mutations, neither the type of alpha chain affected, nor the type of amino acid substituting for glycine, nor the position of the mutation in the alpha chain had a detectable relationship with LS aBMD or histomorphometric results. Thus patients with haploinsufficiency mutations had a milder skeletal phenotype than patients with mutations affecting glycine residues, but there was no clear genotype-phenotype correlation among patients with helical glycine mutations.