Mutation Analysis of COL1A1 and COL1A2 in Fetuses with Osteogenesis Imperfecta Type II/III.

AIM: To analyze COL1A1/2 mutations in prenatal-onset OI for determine the proportion of mutations in type I collagen genes among prenatal onset OI and to provide additional data for genotype-phenotype analyses. MATERIAL AND METHODS: Ten cases of severe fetal short-limb dwarfism detected by antenatal ultrasonography were referred to our center. Before the termination of pregnancy, cordocentesis was performed for fetal karyotype and COL1A1/2 gene sequencing analysis. Postmortem radiographic examination was performed at all instances for definitive diagnosis. RESULTS: COL1A1 and COL1A2 SNP and mutations were identified in all the cases. Among these, one synonymous SNP and four synonymous SNPs were recognized in COL1A1/2, respectively, seven cases have distinct heterozygous mutations and six new COL1A1/2 gene mutations were identified. CONCLUSION: There has been substantial progress in the identification of the molecular defects responsible for skeletal dysplasias. With the constant increase in the number of identified mutations in COL1A1 and COL1A2, genotype-phenotype correlation is becoming increasingly pertinent.

Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta.

During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstntm1Sjl/+) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstntm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2oim), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2oim/+ offspring from natural mating of Mstntm1Sjl/+ dams to Col1a2oim/+sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2oim/+ dams to Col1a2oim/+ sires. Finally, increased bone biomechanical strength of Col1a2oim/+ offspring that had been transferred into Mstntm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta.

Identification of a de novo fetal variant in osteogenesis imperfecta by targeted sequencing-based noninvasive prenatal testing.

Noninvasive prenatal testing (NIPT), which involves analysis of circulating cell-free fetal DNA (cffDNA) from maternal plasma, is highly effective for detecting feto-placental chromosome aneuploidy. However, recent studies suggested that coverage-based shallow-depth NIPT cannot accurately detect smaller single or multi-loci genetic variants. To assess the fetal genotype of any locus using maternal plasma, we developed a novel genotyping algorithm named pseudo tetraploid genotyping (PTG). We performed paired-end captured sequencing of the plasma cell-free DNA (cfDNA), in which case a phenotypically healthy woman is suspected to be carrying a fetus with genetic defect. After a series of independent filtering of 111,407 SNPs, we found one variant in COL1A1 graded with high pathogenic potential which might cause osteogenesis imperfecta (OI). Then, we verified this mutation by Sanger sequencing of fetal and parental blood cells. In addition, we evaluated the accuracy and detection rate of the PTG algorithm through direct sequencing of the genomic DNA from maternal and fetal blood cells. Collectively, our study developed an intuitive and cost-effective method for the noninvasive detection of pathogenic mutations, and successfully identified a de novo variant in COL1A1 (c.2596 G > A, p.Gly866Ser) in the fetus implicated in OI.

Diagnosis of fetal osteogenesis imperfecta by multidisciplinary assessment: a retrospective study of 10 cases.

OBJECTIVE: To describe our 2 year experience in diagnosing prenatal-onset osteogenesis imperfecta (OI) by multidisciplinary assessment. METHODS: We retrospectively analyzed 10 cases of fetal OI by using prenatal ultrasound evaluation, postnatal radiographic diagnosis, and molecular genetic testing of COL1A1/2. RESULTS: By postnatal radiographic examination, five patients were diagnosed with type II OI and five were diagnosed with type III OI. A causative variant in the COL1A1 gene was found in four cases of type II and one case of type III OI; a causative variant in the COL1A2 gene was found in two cases of type III OI. CONCLUSION: The definitive diagnosis of fetal OI should be accomplished using a multidisciplinary assessment, which is paramount for proper genetic counseling. With the discovery of COL1A1/2 gene variants as a cause of OI, sequence analysis of these genes will add to the diagnostic process.

Inducible transient expression of Smpd3 prevents early lethality in fro/fro mice.

Sphingomyelin phosphodiesterase 3 (SMPD3) is a pleiotropic lipid metabolizing enzyme involved in multiple physiological processes. A deletion mutation in the murine Smpd3 gene called fragilitas ossium (fro) leads to severe skeletal abnormalities in the developing fro/fro embryos. Although fro/fro mice can be useful to study many different aspects of SMPD3 functions, their perinatal lethality makes it difficult to generate a sufficient number of mice for controlled studies. In fact, on the C57BL/6 genetic background, none of the fro/fro mice survive beyond the perinatal stage. In this study, we used the "Tet-On" inducible gene expression system to express Smpd3 transiently in fro/fro;ROSA-rtTA;TRE-Smpd3 embryos on the C57BL/6 background. This induced Smpd3 expression corrected all the skeletal abnormalities in these embryos and prevented their early death. However, induction of Smpd3 expression in the adolescent fro/fro;ROSA-rtTA;TRE-Smpd3 mice was not sufficient to correct the defects in trabecular bone mineralization and the impaired growth of the long bones. This novel mouse model will be a useful tool to study SMPD3 biology in vivo.

[Fetal therapy of skeletal dysplasias].

Three types of fetal therapy of skeletal dysplasias, as enzyme replacement, in utero stem cell transplantation, and gene therapy, are reviewed. Clinical trial of recombinant ALP for infantile hypophosphatasia has already started in Japan. In future, such enzyme replacement therapy is expected to be adapted to fetus. There are several reports of mesenchymal stem cell transplantation for osteogenesis imperfecta fetus. These case reports have showed that stem cell transplantation is safe and to some extent works in patients. No clinical trial for gene therapy has been reported. Recently, the study of gene therapy of using HPP fetal mouse showed an excellent therapeutic effect. Fetal therapy of skeletal dysplasias is still the stage of research because of the safety and the ethical issues. However, in order to treat severe cases of skeletal dysplasias which abnormal development has been already completed at birth, fetal therapy at an early stage would be demanded.

Mutation and polymorphism spectrum in osteogenesis imperfecta type II: implications for genotype-phenotype relationships.

Osteogenesis imperfecta (OI), also known as brittle bone disease, is a clinically and genetically heterogeneous disorder primarily characterized by susceptibility to fracture. Although OI generally results from mutations in the type I collagen genes, COL1A1 and COL1A2, the relationship between genotype and phenotype is not yet well understood. To provide additional data for genotype-phenotype analyses and to determine the proportion of mutations in the type I collagen genes among subjects with lethal forms of OI, we sequenced the coding and exon-flanking regions of COL1A1 and COL1A2 in a cohort of 63 subjects with OI type II, the perinatal lethal form of the disease. We identified 61 distinct heterozygous mutations in type I collagen, including five non-synonymous rare variants of unknown significance, of which 43 had not been seen previously. In addition, we found 60 SNPs in COL1A1, of which 17 were not reported previously, and 82 in COL1A2, of which 18 are novel. In three samples without collagen mutations, we found inactivating mutations in CRTAP and LEPRE1, suggesting a frequency of these recessive mutations of approximately 5% in OI type II. A computational model that predicts the outcome of substitutions for glycine within the triple helical domain of collagen alpha1(I) chains predicted lethality with approximately 90% accuracy. The results contribute to the understanding of the etiology of OI by providing data to evaluate and refine current models relating genotype to phenotype and by providing an unbiased indication of the relative frequency of mutations in OI-associated genes.

Pamidronate Administration During Pregnancy and Lactation Induces Temporal Preservation of Maternal Bone Mass in a Mouse Model of Osteogenesis Imperfecta.

During pregnancy and lactation, the maternal skeleton undergoes significant bone loss through increased resorption to provide the necessary calcium supply to the developing fetus and suckling neonate. This period of skeletal vulnerability has not been clearly associated with increased maternal fracture risk, but these physiological conditions can exacerbate an underlying metabolic bone condition like osteogenesis imperfecta. Although bisphosphonates (BPs) are commonly used in postmenopausal women, there are cases where premenopausal women taking BPs become pregnant. Given BPs' long half-life, there is a need to establish how BPs affect the maternal skeleton during periods of demanding metabolic bone changes that are critical for the skeletal development of their offspring. In the present study, pamidronate- (PAM-) amplified pregnancy-induced bone mass gains and lactation-induced bone loss were prevented. This preservation of bone mass was less robust when PAM was administered at late stages of lactation compared with early pregnancy and first day of lactation. Pregnancy-induced osteocyte osteolysis was also observed and was unaffected with PAM treatment. No negative skeletal effects were observed in offspring from PAM-treated dams despite lactation-induced bone loss prevention. These findings provide important insight into (1) a treatment window for when PAM is most effective in preserving maternal bone mass, and (2) the maternal changes in bone metabolism that maintain calcium homeostasis crucial for fetal and neonatal bone development. © 2019 American Society for Bone and Mineral Research.

Enzyme histochemical localisation of alkaline phosphatase activity in osteogenesis imperfecta bone and growth plate: a preliminary study.

At the ultrastructural level alkaline phosphatase has been studied in calcifying cartilage but not in bone. The aim of this study was to assess if there is an osteoblast dysfunction in Osteogenesis Imperfecta (OI) with respect to alkaline phosphatase activity. Specimens from three OI type II foetal femoral bones, two OI type II growth plates, one normal foetal femoral bone and growth plate, one OI type III femoral bone specimen and one normal juvenile bone specimens were examined using modified lead nitrate method to identify alkaline phosphatase reactivity. The electron dense reaction product (indicative of the presence of alkaline phosphatase) was demonstrable on the cell membrane of the osteoblasts, as focal concentrations in the collagen osteoid and on the mineralisation front of normal bone. In normal bone the intensity of the reaction seemed to be stronger than in OI bone and appeared as a continuous black line along the osteoblast cell membranes. In OI bone the reaction product only appeared as a few electron dense beads along the osteoblast cell membrane. There appeared to be reduced and diffuse reaction product on OI osteoblasts, thus implying either a reduced level and/or altered activity of alkaline phosphatase and hence a dysfunction of osteoblasts. This confirms the findings of the previous report of the impaired activity of alkaline phosphatase in OI osteoblasts. Even in the OI growth plate, hypertrophic chondrocytes showed less intense reaction product than the chondrocytes in the normal growth plate. The normal human growth plates used in this study showed a similar pattern, but in the OI growth plate even the hypertrophic zone, where the alkaline phosphatase activity is reported to be high, showed less intense reaction product. Biochemical reports indicate that alkaline phosphatase levels are normal in cultured OI cell lines, yet ultrastructural histochemical observations reported here, show reduced enzyme localisation and this may suggest reduced amounts of protein or reduced activity at the tissue level.

A case of fetal osteogenesis imperfecta type 2A: longitudinal observation of natural course in utero and pitfalls for prenatal ultrasound diagnosis.

We present a case of osteogenesis imperfecta (OI) type 2A in which a natural course in utero was observed from 23 weeks' gestation to term. At 23 weeks' gestation, a sonographic examination showed a cloverleaf skull-like head, a narrow thorax, and marked shortening of the long bones with bowing of the femurs and humeri. Follow-up examinations showed that the cloverleaf skull-like head was not evident at 28 weeks' gestation. Discontinuity of the ribs and femurs was observed at 26 and 30 weeks' gestation, respectively. This finding suggested bone fractures, which were confirmed by three-dimensional computed tomography at 32 weeks' gestation. Ultrasonographic findings of bones, including the long bones and calvarium, changed with advancing gestation during the second trimester. Characteristic features of OI type 2A were evident during the late second to early third trimesters. Repeated ultrasonographic examinations together with three-dimensional computed tomography are necessary for the definitive diagnosis of OI type 2A in the second trimester.

COL1A2 and COL2A1 expression in temporal bone of lethal osteogenesis imperfecta.

OBJECTIVE: COL1A2 and COL2A1 genes are expressed at high levels in many cochlear cells of 16- to 23-week-old human fetuses. Given these prior observations and the rare opportunity to obtain temporal bones from a deceased neonate with osteogenesis imperfecta (OI) type II, we determined the cellular distribution and level of expression of COL1A2 mRNA in OI type II inner ear compared with the expression in second-trimester human fetal cochlea. Expression of COL2A1 mRNA was assessed for its normal role in OI type II neonatal cochlea and to address potential spatial and temporal changes along with our observations in fetal cochlea. We describe our tissue in situ hybridization protocol and document its usefulness in assessing gene expression in human temporal bone obtained at autopsy. DESIGN: RNA-RNA in situ hybridization was performed in formaldehyde-fixed, decalcified, paraffin-embedded temporal bone sections from a neonate with OI type II. Semi-quantitative assessment of gene expression was performed by visual inspection of grain densities. RESULTS AND CONCLUSIONS: COL1A2 and COL2A1 were expressed at moderate-to-high levels in many membranous cochlear cells, and no dramatic alterations in pattern or level of expression of these genes was noted compared with human fetal cochlea. Consistent with in vitro studies, expression of COL1A2 in osteoblasts lining enchondral and endosteal layers is less than that in identical cells of the fetal otic capsule undergoing osteoid deposition and mineralization. Expression of COL1A2 mRNA in osteoblasts lining the outer periosteum of otic capsule is markedly higher than osteoblasts lining enchondral and endosteal layers, suggesting that differential expression may exist between osteoblasts lining endosteal, enchondral, and periosteal surfaces of bone in OI type II.

Initial experience using magnetic resonance imaging in prenatal diagnosis of osteogenesis imperfecta type II: a case report.

We report a fetus with osteogenesis imperfecta (OI) first diagnosed by ultrasound in routine prenatal examination and further evaluated by magnetic resonance imaging (MRI). Fetal MRI was undertaken with a 1.5-T magnet using a body-phased array coil and an ultrafast imaging technique, half-Fourier single-shot turbo spin-echo (HASTE). Radiological examination shortly after birth and postmortem examination confirmed the prenatal diagnosis. In this case, fetal MRI provided excellent spatial and tissue resolution with multiplanar display. It revealed additional diagnostic information and improved imaging conspicuity. MRI complemented sonography for further differentiating clinical and sonographic findings.

[The temporomandibular joint (TMJ) and osteogenesis imperfecta. A study of its embryologic development]. / Articolazione temporomandibolare (ATM) ed osteogenesi imperfetta. Studio sullo sviluppo embriologico.

Although the most common oral manifestation of osteogenesis imperfecta is dentinogenesis imperfecta, several authors have described jaw fractures and radiolucent bone lesions associated with this disease. The authors, after reporting the main features of this disease, compare the cranial embryologic growth in twice fetuses of a 15 weeks old: one of these with osteogenesis imperfecta.

[Significance of the length of the fetal femur]. / A magzati femur hosszmérésének jelentösége.

The importance of the examination of fetal skeletal system, especially the measurement of femur length must be emphasized during the routine ultrasound screening. A case of osteogenesis imperfecta is described, diagnosed on the 18th week of pregnancy by ultrasound.

[Increased nuchal translucency in osteogenesis imperfecta]. / Forstørret nakkefold kan ses ved osteogenesis imperfecta.

A limited number of reports published since 2001 have described an association between increased nuchal translucency (NT) and osteogenesis imperfecta (OI). We report a new case which underlines the frequency of this association as well as the importance of follow-up and genetic evaluation. In the present case, ultrasound scanning at 13 weeks of gestation showed a NT of 3.2 mm and no other pathological findings. At 20 weeks a severe skeletal dysplasia was diagnosed by ultrasound. The pathology report of the aborted foetus indicated OI, and DNA analysis confirmed a COL1A1 mutation.

Fetus with osteogenesis imperfecta presenting as increased nuchal translucency thickness in the first trimester.

Type II osteogenesis imperfecta (OI) is a perinatally lethal disorder due to type I collagen abnormalities that has been diagnosed successfully in the second trimester. We report a case of type II OI that was confirmed on postmortem histology and radiography presenting as increased nuchal translucency in the first trimester.

Osteogénesis imperfecta tipo II: estudio de dos casos / Osteogenesis imperfecta type II: report of two cases

Se presentan dos casos clínicos de fetos con osteogénesis imperfecta tipo II, productos de partos vaginales de pretérmino, de sexo femenino, ambos sin signos vitales y con malformaciones congénitas evidentes desde el punto de vista esquelético. En ambos fetos se evidencia al estudio macroscópico cráneo blando y aplastado, colapso de la región tóracoabdominal, acortamiento de las extremidades y genitales externos femeninos. En la disección se aprecia hipoplasia pulmonar y malrotación intestinal. El estudio radiológico evidencia ausencia total de osificación para el primer feto y solo puntos rudimentarios para la base del cráneo, omóplato, húmero, un hueso del antebrazo, el ilíaco, el fémur y un hueso de la pierna, para el segundo feto. El tratamiento actual del que se dispone está muy lejos de ser exitoso y se restringe a terapia ortopédica y administración de bifosfonatos. El trasplante de células mesenquimáticas y su transformación a células osteogénicas, abre nuevas perspectivas en el manejo de la enfermedad

We present two clinical cases of fetuses with osteogenesis imperfecta type II, products of preterm vaginal deliveries, both females, without vital signs and congenital malformations evident in the skeletal system. Macroscopical study shows in both fetuses soft and crushed skull, collapse of the thoracoabdominal region, shortening of the limbs and external female genitalia. Pulmonary hypoplasia and intestinal malrotation are evident at the dissection. Radiological screening shows total absence of ossification for the first fetus and only rudimentary points for the base of the skull, scapula, humerus, a forearm bone, the ilium, femur and leg bon for the second fetus. Current treatment available is far from being successful and is limited to orthopedic therapy and administration of bisphosphonates. Transplantation of mesenchymal cells and their transformation into osteogenic cells, opens new perspectives on the management of the disease

Prenatal diagnosis of Bruck syndrome.

Bruck syndrome is an autosomal recessive connective tissue disorder combining features of osteogenesis imperfecta and arthrogryposis multiplex congenita. There are only few reports describing this rare syndrome of multiple fractures and joint contractures that is thought to be a subtype of osteogenesis imperfecta. We report the first case of prenatal diagnosis of this syndrome in a fetus at 23 weeks of gestation. Ultrasound findings included brachycephaly, retrognathia marked shortening and bowing of both femurs, bilateral fixed flexion of the elbows, bilateral fixed extension of the wrists and partially fixed flexion of the knees. The parents opted for termination of pregnancy. Macroscopic and radiologic examination of the aborted fetus confirmed the prenatal diagnosis, whereas morphological studies of the bone tissue found no hard evidence of osteogenesis imperfecta, probably due to the early stage of pregnancy and the heterogeneity of the syndrome itself.

An ultrastructural and immunogold localization study of proteoglycans associated with the osteocytes of fetal bone in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a rare, heterogeneous, inherited connective tissue disorder frequently caused by abnormalities of type I collagen. It is characterized by bone fragility, osteopenia, and progressive skeletal deformities. Electron microscopy of three OI type II fetal bone samples revealed numerous large osteocyte lacunae. In addition, there was a perilacunar osteoid-like band of collagen surrounding the osteocytes, which was unmineralized and morphologically unusual. Furthermore, large osteocyte lacunae contained fine particles and filamentous material similar to the expected ultrastructural appearance of proteoglycans. More detailed examination was carried out using histochemical and immunogold localization of proteoglycans at light and ultrastructural levels. These tests and the use of electron probe X-ray microanalysis confirmed that the material in the osteocyte lacunae was proteoglycan. In contrast, in the age- and site-matched normal fetal bone, all the osteocyte lacunae appeared negative for proteoglycan. Proteoglycans are regarded as inhibitors of calcification. Our observation of substantial amounts of proteoglycan in abnormally enlarged osteocytic lacunae of some OI fetal bone suggests association with the abnormal bone of this particular subtype of OI type II.