Two novel distinct COL1A2 mutations highlight the complexity of genotype–phenotype correlations in osteogenesis imperfecta and related connective tissue disorders

Osteogenesis imperfecta is a heritable connective tissue disorder characterized by variable symptoms including predisposition to fractures. Despite the identification of numerous mutations, a reliable genotype–phenotype correlation has remained notoriously difficult. We now describe two patients with osteogenesis imperfecta and novel, so far undescribed mutations in the COL1A2 gene, further highlighting this complexity. A 3-year-old patient presented with features reminiscent of a connective tissue disorder, with joint hypermobility, Wormian bones, streaky lucencies in the long bones and relative macrocephaly. The patient carried a heterozygous c.1316G > A (p.Gly439Asp) mutation in the COL1A2 gene located in a triple-helix region, in which glycine substitutions have been assumed to cause perinatal lethal OI (Sillence type II). A second family with type I osteogenesis imperfecta carried a heterozygous nonsense mutation c.4060C > T (p.Gln1354X) within the last exon of COL1A2. Whereas other heterozygous nonsense mutations in COL1A2 do not lead to a phenotype, in this case the mRNA is presumed to escape nonsense-mediated decay. Therefore the predicted COL1A2 propeptide lacks the last 13 C-terminal amino acids, suggesting that the OI phenotype results from decelerated assembly and overmodification of the collagen triple helix. The presented COL1A2 mutations exemplify the complexity of COL1A2 genotype–phenotype correlation in genetic counselling in OI.     

Genotype�Cphenotype 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 α1 or the α2 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 α1(I) (n=67) or α2(I) (n=94). Serine substitutions were the most frequently encountered type of mutation in both chains. Compared with patients with serine substitutions in α2(I) (n=40), patients with serine substitutions in α1(I) (n=42) on average were shorter (median height z-score −6.0 vs −3.4; P=0.005), indicating that α1(I) mutations cause a more severe phenotype. Height correlated with the location of the mutation in the α2(I) chain but not in the α1(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.     

G76E Substitution in Type I Collagen Is the First Nonlethal Glutamic Acid Substitution in the α1(I) Chain and Alters Folding of the N-terminal End of the Helix

The majority of osteogenesis imperfecta (OI) is caused by substitutions for glycine residues in the two α chains of type I collagen. Since only 4% of possible nucleotide changes in type I collagen glycine codons would result in a glutamic acid substitution, these are predicted to be infrequent. Only one glutamic acid substitution in type I collagen has been fully reported. We describe here the clinical, biochemical, and molecular characterization of a girl with severe type III OI caused by a G76E substitution in COL1A1. This is the first delineation of a glutamic acid substitution in the α1(I) chain causing nonlethal osteogenesis imperfecta. The proband's fibroblast type I collagen chains and cyanogen bromide peptides were electrophoretically normal, while osteoblast collagen was slightly overmodified. This suggested a mutation near the N-terminal end of the collagen helix. A mismatch was detected by RNA:DNA hybrid analysis in cDNA coding for 106 amino acids at the N-terminal end of the helical region. Subclones of both alleles were sequenced and revealed a G → A (c.761G > A) mutation causing an α1(I) G76E substitution in one allele. The presence of the mutation in the proband's leukocyte gDNA, and its absence in parental gDNA, was confirmed by Tsp509I digestion. The glutamic acid substitution alters the folding of the mutant collagen helices. Pericellular processing of type I collagen by the proband's fibroblasts yielded an earlier appearance of the pC-α1(I) form and of mature α chains as compared to control cell processing. Also, the presence of the glutamic acid substitution apparently exposes the adjacent Arg75 residue in the α1 chain. Trypsin digestion of proband fibroblast collagen resulted in shortened α1 chains, as confirmed by CNBr analysis. In addition, the Tm for mutant helices from fibroblasts and osteoblasts was decreased 2–4°C versus controls, demonstrating a decrease in helix stability. These findings increase our understanding of the disruptive effect of glutamic acid substitutions in collagen.     

Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a mutation in the COL1A2 gene of type I collagen. The mosaic parent exhibits phenotypic features of a mild form of the disease.

We have determined that a man, ascertained because he fathered a child with lethal osteogenesis imperfecta (OI) with each of two partners, is mosaic in both his germline and somatic tissues for a mutation in the COL1A2 gene which encodes the pro alpha 2(I) chain of type I procollagen. His dermal fibroblasts were previously shown to synthesize a population of cysteine-containing alpha 2(I) chains that were posttranslationally overmodified. DNA sequence analysis of COL1A2 cDNAs demonstrated that the cysteine-containing chain resulted from a point mutation (G to T) in the first position of the codon for the glycine at residue 472 of the triple helical domain. Genomic DNA from the one available affected infant contained the mutant and normal COL1A2 alleles in equal proportion. Examination of DNA from several tissues of the father showed that the mutant allele was present in approximately 40% of his sperm, 80% of his lymphocytes, and nearly 100% of his dermal fibroblasts. Despite the high level of mosaicism detected in somatic tissues, the only phenotypic manifestation of OI in the proband was that he was shorter than his unaffected male relatives and had mild dentinogenesis imperfecta. Thermal stability of type I collagen molecules containing the substitution was decreased, but to a lesser extent than for a nonlethal cysteine for glycine substitution at residue 259 of alpha 2(I), indicating that this measure of molecular stability may be of limited use in explaining the pathogenesis of osteogenesis imperfecta.     

Genetic epidemiology,prevalence, and genotype–phenotype correlations in the Swedish population with osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a rare hereditary bone fragility disorder, caused by collagen I mutations in 90% of cases. There are no comprehensive genotype–phenotype studies on >100 families outside North America, and no population-based studies determining the genetic epidemiology of OI. Here, detailed clinical phenotypes were recorded, and the COL1A1 and COL1A2 genes were analyzed in 164 Swedish OI families (223 individuals). Averages for bone mineral density (BMD), height and yearly fracture rate were calculated and related to OI and mutation type. N-terminal helical mutations in both the α1- and α2-chains were associated with the absence of dentinogenesis imperfecta (P<0.0001 vs 0.0049), while only those in the α1-chain were associated with blue sclera (P=0.0110). Comparing glycine with serine substitutions, α1-alterations were associated with more severe phenotype (P=0.0031). Individuals with type I OI caused by qualitative vs quantitative mutations were shorter (P<0.0001), but did not differ considering fractures or BMD. The children in this cohort were estimated to represent >95% of the complete Swedish pediatric OI population. The prevalence of OI types I, III, and IV was 5.16, 0.89, and 1.35/100 000, respectively (7.40/100 000 overall), corresponding to what has been estimated but not unequivocally proven in any population. Collagen I mutation analysis was performed in the family of 97% of known cases, with causative mutations found in 87%. Qualitative mutations caused 32% of OI type I. The data reported here may be helpful to predict phenotype, and describes for the first time the genetic epidemiology in >95% of an entire OI population.     

G76E substitution in type I collagen is the first nonlethal glutamic acid substitution in the alpha1(I) chain and alters folding of the N-terminal end of the helix

The majority of osteogenesis imperfecta (OI) is caused by substitutions for glycine residues in the two alpha chains of type I collagen. Since only 4% of possible nucleotide changes in type I collagen glycine codons would result in a glutamic acid substitution, these are predicted to be infrequent. Only one glutamic acid substitution in type I collagen has been fully reported. We describe here the clinical, biochemical, and molecular characterization of a girl with severe type III OI caused by a G76E substitution in COL1A1. This is the first delineation of a glutamic acid substitution in the alpha1(I) chain causing nonlethal osteogenesis imperfecta. The proband's fibroblast type I collagen chains and cyanogen bromide peptides were electrophoretically normal, while osteoblast collagen was slightly overmodified. This suggested a mutation near the N-terminal end of the collagen helix. A mismatch was detected by RNA:DNA hybrid analysis in cDNA coding for 106 amino acids at the N-terminal end of the helical region. Subclones of both alleles were sequenced and revealed a G --> A (c.761G > A) mutation causing an alpha1(I) G76E substitution in one allele. The presence of the mutation in the proband's leukocyte gDNA, and its absence in parental gDNA, was confirmed by Tsp509I digestion. The glutamic acid substitution alters the folding of the mutant collagen helices. Pericellular processing of type I collagen by the proband's fibroblasts yielded an earlier appearance of the pC-alpha1(I) form and of mature alpha chains as compared to control cell processing. Also, the presence of the glutamic acid substitution apparently exposes the adjacent Arg75 residue in the alpha1 chain. Trypsin digestion of proband fibroblast collagen resulted in shortened alpha1 chains, as confirmed by CNBr analysis. In addition, the Tm for mutant helices from fibroblasts and osteoblasts was decreased 2-4 degrees C versus controls, demonstrating a decrease in helix stability. These findings increase our understanding of the disruptive effect of glutamic acid substitutions in collagen.     

Prenatal prediction of osteogenesis imperfecta (OI type IV): exclusion of inheritance using a collagen gene probe.

Autosomal dominant osteogenesis imperfecta is caused by mutations in the COL1A2 and COL1A1 genes of type I collagen. In a family with OI type IV genetically linked to the COL1A2 gene, we attempted prenatal diagnosis in a pregnancy at risk by genotyping the DNA of the fetus for a COL1A2 gene associated RFLP. Our results showed that the fetus inherited the normal COL1A2 allele from her affected parent. Linkage analysis can thus be used in the prenatal diagnosis of dominantly inherited osteogenesis imperfecta.     

Mutational spectrum of type I collagen genes in Korean patients with osteogenesis imperfecta

Mutations in the type I collagen genes COL1A1 and COL1A2 are responsible for the dominantly inherited connective tissue disorder osteogenesis imperfecta (OI). The severity of OI is diverse, ranging from perinatal lethality to a very mild phenotype that is characterized by normal stature and the absence of deformities. Although there have been several studies on the mutational spectra of COL1A1 and/or COL1A2 in Western populations, very few cases have been reported from Asia. In this study, we investigated 67 unrelated Korean probands with OI and used nucleotide sequence analysis to detect COL1A1 and COL1A2 mutations. Thirty-five different mutations were identified in the two genes, including 24 novel mutations. Among the 35 kinds of detected mutations, 15 were glycine substitutions (seven in COL1A1 and eight in COL1A2), one was a nonsense mutation, four were frameshift mutations in COL1A1, three were in-frame duplications in COL1A2, and 12 were splice site mutations (seven in COL1A1 and five in COL1A2). Until now, mutations in the COL1A1 and COL1A2 genes known to cause OI were unique and rarely repeated in other families. Interestingly, the c.982G>A (p.Gly328Ser) mutation in COL1A2 was found recurrently and was the causative mutation in five independent OI probands. Haplotype analysis of the COL1A2 gene revealed that four probands from five independent OI probands with c.982G>A (p.Gly328Ser) had a common haplotype. Our clinical data showed the heterogeneity even within a specific genotype, which suggested the complex expression of this disease.     

Mutations in the COL1A2 gene of type I collagen that result in nonlethal forms of osteogenesis imperfecta

Although virtually all mutations that result in osteogenesis imperfecta (OI) affect the genes that encode the chains of type I procollagen, the effects of mutations in the COL1A2 gene have received less attention than those in the COL1A1 gene. We have characterized mutations in 4 families that give rise to different OI phenotypes. In three families substitutions of glycine residues by cysteine in the triple helical domain (a single example at position 259 and 2 families in which substitution of glycine at 646 by cysteine) have been identified, and in the fourth a G for A transition at position + 4 in intron 33 led to use of an alternative splice site and inclusion of 6 amino acids (val-gly-arg-ile-leu-phe) between residues 585 and 586 of the normal triple helix. The relation between position of substitution of glycine by cysteine in the COL1A2 gene does not follow the pattern developed in the COL1A1 gene. To determine how COL1A2 mutations produce OI phenotypes, we have produced a full-length mouse cDNA into which we plan to place mutations and examine their effects in stably transfected osteogenic cells and in transgenic animals.     

Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels - a Novel Therapeutic Approach in Osteogenesis Imperfecta

Osteogenesis imperfecta, also known as “brittle bone disease”, is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3''UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3''UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis imperfecta.     

Haplotype analysis of collagen type I genes in the general population and in osteogenesis imperfecta families

The allele frequencies of 2 new polymorphic markers of collagen type I proalpha 1 (COL1A1) and proalpha 2 (COL1A2) genes were determined in a random sample of chromosomes by polymerase chain reaction. The minor allele frequencies were 0.27 for COL1A1/+88Mn1I, and 0.39 for COL1A2/1446 PvuII RFLPs, respectively. These 2 polymorphisms increased the combined (PIC) values we previously determined in the Italian population with Southern blotting procedures, from 0.71 at the COL1A1 locus to 0.81, and from 0.71 at the COL1A2 locus to 0.88, respectively. With a combination of these markers, we have carried out the segregation analysis of 4 new families in which osteogenesis imperfecta (OI) segregated as a dominant trait. The disease segregated with COL1A1 in 2 OI type I families, and with COL1A2 in one OI type IV family. In one OI type I family the concordant locus was uncertain. This analysis was extended to the 7 dominant OI families we previously reported: in 3 out of 11 pedigrees either locus still could not be excluded, indicating the need for more genetic markers. COL1A1 and COL1A2 haplotype frequencies were compared in normal and OI chromosomes: no preferential association of the disease with a given haplotype was detected. The correlation between affected locus and clinical aspects is discussed.     

A single amino acid substitution (D1441Y) in the carboxyl-terminal propeptide of the proalpha1(I) chain of type I collagen results in a lethal variant of osteogenesis imperfecta with features of dense bone diseases

Osteogenesis imperfecta (OI) is characterised by brittle bones and caused by mutations in the type I collagen genes, COL1A1 and COL1A2. We identified a mutation in the carboxyl-terminal propeptide coding region of one COL1A1 allele in an infant who died with an OI phenotype that differed from the usual lethal form and had regions of increased bone density. The newborn female had dysmorphic facial features, including loss of mandibular angle. Bilateral upper and lower limb contractures were present with multiple fractures in the long bones and ribs. The long bones were not compressed and their ends were radiographically dense. She died after a few hours and histopathological studies identified extramedullary haematopoiesis in the liver, little lamellar bone formation, decreased osteoclasts, abnormally thickened bony trabeculae with retained cartilage in long bones, and diminished marrow spaces similar to those seen in dense bone diseases such as osteopetrosis and pycnodysostosis. The child was heterozygous for a COL1A1 4321G→T transversion in exon 52 that changed a conserved aspartic acid to tyrosine (D1441Y). Abnormal proα1(I) chains were slow to assemble into dimers and trimers, and abnormal molecules were retained intracellularly for an extended period. The secreted type I procollagen molecules synthesised by cultured dermal fibroblasts were overmodified along the full length but had normal thermal stability. These findings suggest that the unusual phenotype reflected both a diminished amount of secreted type I procollagen and the presence of a population of stable and overmodified molecules that might support increased mineralisation or interfere with degradation of bone.     

Biochemical screening of type I collagen in osteogenesis imperfecta: detection of glycine substitutions in the amino end of the alpha chains requires supplementation by molecular analysis

Background

The biochemical test for osteogenesis imperfecta (OI) detects structural abnormalities in the helical region of type I collagen as delayed electrophoretic migration of alpha chains on SDS‐urea‐PAGE. Sensitivity of this test is based on overmodification of alpha chains in helices with a glycine substitution or other structural defect. The limits of detectability have not been reported.

Methods

We compared the collagen electrophoretic migration of 30 probands (types III or IV OI) with known mutations in the amino half of the α1(I) and α2(I) chains. Differences in sensitivity were examined by 5% and 6% SDS‐urea‐PAGE, and with respect to alpha chain, location along the chain, and substituting amino acid.

Results

Sensitivity was enhanced on 5% gels, and by examination of intracellular and secreted collagen. In α1(I), substitutions in the first 100 residues were not detectable; 7% of cases in the current Mutation Consortium database are in this region. α1(I) substitutions between residues 100 and 230 were variably detectable, while those after residue 232 were all detected. In α2(I), variability of electrophoretic detection extended through residue 436. About a third of cases in the Consortium database are located in the combined variable detection region. Biochemical sensitivity did not correlate with substituting residue.

Conclusions

Complete testing of probands with normal type I collagen biochemical results requires supplementation by molecular analysis of cDNA or gDNA in the amino third of α1(I) and amino half of α2(I). Mutation detection in OI is important for counselling, reproductive decisions, exclusion of child abuse, and genotype‐phenotype correlations.     

Osteogenesis imperfecta type III: mutations in the type I collagen structural genes, COL1A1 and COL1A2, are not necessarily responsible.

Most forms of osteogenesis imperfecta are caused by dominant mutations in either of the two genes, COL1A1 and COL1A2, that encode the pro alpha 1(I) and pro alpha 2(I) chains of type I collagen, respectively. However, a severe, autosomal recessive form of OI type III with a comparatively high frequency has been recognised in the black populations of southern Africa. We preformed linkage analyses in eight OI type III families using RFLPs associated with the COL1A1 and COL1A2 loci to determine whether mutations in the genes for type I collagen were responsible for this form of OI. Recombination between the OI phenotype and polymorphic markers at both loci was shown in three of the eight families investigated. The combined lod scores for the eight families were -10.6 for COL1A1 and -11.2 for COL1A2. Further, we examined the type I procollagen produced by skin fibroblast cultures derived from 15 affected and 12 unaffected subjects from the above eight families plus one further family. We found no evidence for defects in the synthesis, structure, secretion, or post-translational modification of the chains of type I procollagen produced by any of the family members. These results suggest that mutations within or near the type I collagen structural genes are not responsible for this form of OI.     

Disruption of one intra-chain disulphide bond in the carboxyl-terminal propeptide of the proα1(I) chain of type I procollagen permits slow assembly and secretion of overmodified, but stable procollagen trimers and results in mild osteogenesis imperfecta

Type I procollagen is a heterotrimer comprised of two proα1(I) chains and one proα2(I) chain. Chain recognition, association, and alignment of proα chains into correct registration are thought to occur through interactions between the C-terminal propeptide domains of the three chains. The C-propeptide of each chain contains a series of cysteine residues (eight in proα1(I) and seven in proα2(I)), the last four of which form intra-chain disulphide bonds. The remaining cysteine residues participate in inter-chain stabilisation. Because these residues are conserved, they are thought to be important for folding and assembly of procollagen. We identified a mutation (3897C→G) that substituted tryptophan for the cysteine at position 1299 in proα1(I) (C1299W, the first cysteine that participates in intra-chain bonds) and resulted in mild osteogenesis imperfecta. The patient was born with a fractured clavicle and four rib fractures. By 18 months of age he had had no other fractures and was on the 50th centile for length and weight. The proband's mother, maternal aunt, and grandfather had the same mutation and had few fractures, white sclerae, and discoloured teeth, but their heights were within the normal range. In the patient's cells the defective chains remained as monomers for over 80 minutes (about four times normal) and were overmodified. Some secreted procollagens were also overmodified but had normal thermal stability, consistent with delayed, but normal helix formation. This intra-chain bond may stabilise the C-propeptide and promote rapid chain association. Other regions of the C-propeptide thus play more prominent roles in chain registration and triple helix nucleation.


Keywords: osteogenesis imperfecta; procollagen; mutation; carboxyl-terminal propeptide     

Complete COL1A1 allele deletions in osteogenesis imperfecta

PurposeTo identify a molecular genetic cause in patients with a clinical diagnosis of osteogenesis imperfecta (OI) type I/IV.MethodsThe authors performed multiplex ligation-dependent probe amplification analysis of the COL1A1 gene in a group of 106 index patients.ResultsIn four families with mild osteogenesis imperfecta and no other phenotypic abnormalities, a deletion of the complete COL1A1 gene on one allele was detected, a molecular finding that to our knowledge has not been described before, apart from a larger chromosomal deletion detected by fluorescent in situ hybridization encompassing the COL1A1 gene in a patient with mild osteogenesis imperfecta and other phenotypic abnormalities. Microarray analysis in three of the four families showed that it did not concern a founder mutation.ConclusionThe clinical picture of complete COL1A1 allele deletions is a comparatively mild type of osteogenesis imperfecta. As such, multiplex ligation-dependent probe amplification analysis of the COL1A1 gene is a useful additional approach to defining the mutation in cases of suspected osteogenesis imperfecta type I with no detectable mutation.     

Total absence of the alpha2(I) chain of collagen type I causes a rare form of Ehlers-Danlos syndrome with hypermobility and propensity to cardiac valvular problems

Background

Heterozygous mutations in the COL1A1 or COL1A2 gene encoding the α1 and α2 chain of type I collagen generally cause either osteogenesis imperfecta or the arthrochalasis form of Ehlers‐Danlos syndrome (EDS). Homozygous or compound heterozygous COL1A2 mutations resulting in complete deficiency of the proα2(I) collagen chains are extremely rare and have been reported in only a few patients, albeit with variable phenotypic outcome.

Methods

The clinical features of the proband, a 6 year old boy, were recorded. Analysis of proα and α‐collagen chains was performed by SDS‐polyacrylamide gel electrophoresis using the Laemmli buffer system. Single stranded conformation polymorphism analysis of the proband''s DNA was also carried out.

Results

In this report we show that complete lack of proα2(I) collagen chains can present as a phenotype reminiscent of mild hypermobility EDS during childhood.

Conclusions

Biochemical analysis of collagens extracted from skin fibroblasts is a powerful tool to detect the subset of patients with complete absence of proα2(I) collagen chains, and in these patients, careful cardiac follow up with ultrasonography is highly recommended because of the risk for cardiac valvular problems in adulthood.     

Osteogenesis imperfecta: clinical, biochemical and molecular findings

Mutations in COL1A1 and COL1A2 genes, encoding the alpha1 and alpha2 chain of type I collagen, respectively, are responsible for the vast majority of cases of osteogenesis imperfecta (OI) (95% of patients with a definite clinical diagnosis). We have investigated 22 OI patients, representing a heterogeneous phenotypic range, at the biochemical and molecular level. A causal mutation in either type I collagen gene was identified in 20 of them: no recurrent mutation was found in unrelated subjects; 15 out of 20 mutations had not been reported previously. In two patients, we could not find any causative mutation in either type I collagen gene, after extensive genomic DNA sequencing. Failure of COL1A1/COL1A2 mutation screening may be due, in a few cases, to further clinical heterogeneity, i.e. additional non-collagenous disease loci are presumably involved in OI types beyond the traditional Sillence's classification.     

A novel mutation combining with rs66612022 in a Chinese pedigree suggests a new pathogenesis to osteogenesis imperfecta via whole genome sequencing

Osteogenesis imperfecta (OI) is a rare heritable disease with systemic connective tissue disorder. Most of the patients represent autosomal dominant form of OI, and are usually resulting from the mutations in type I collagen genes. However, the gene mutations reported previously only account for ∼70% of the OI cases. Here, in a Chinese OI family, we examined seven patients and nine normal individuals using the whole genome sequencing and molecular genetic analysis. The mutation of rs66612022 (COL1A2:p.Gly328Ser) related to glycine substitution was found in the seven patients. Moreover, we identified a novel missense mutation (HMMR:p.Glu2Gln). Interestingly, the individuals of this family with both the mutations were suffering from OI, while the others carried one or none of them are normal. The mutations of COL1A2 and HMMR and their combined effect on OI would further expand the genetic spectrum of OI.