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1.  Peptidyl 3-Hydroxyproline-Binding Properties of Type I Collagen Suggest a Function in Fibril Supramolecular Assembly 
Biochemistry  2012;51(12):2417-2424.
Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, 3S, 2S-L-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss or reduction of 3-hydroxylation at two proline residues, α1(I) Pro986 and α2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism to explain the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the α1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study on the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.
doi:10.1021/bi2019139
PMCID: PMC3314591  PMID: 22380708
collagen; 3-hydroxyproline; bone; supramolecular assembly; osteogenesis imperfecta
2.  Dominant and Recessive Forms of Fibrochondrogenesis Resulting from Mutations at a Second Locus, COL11A2 
Fibrochondrogenesis is a severe, recessively inherited skeletal dysplasia shown to result from mutations in the gene encoding the proα1(XI) chain of type XI collagen, COL11A1. The first of two cases reported here was the affected offspring of first cousins and sequence analysis excluded mutations in COL11A1. Consequently, whole-genome SNP genotyping was performed to identify blocks of homozygosity, identical-by-descent, wherein the disease locus would reside. COL11A1 was not within a region of homozygosity, further excluding it as the disease locus, but the gene encoding the proα2(XI) chain of type XI collagen, COL11A2, was located within a large region of homozygosity. Sequence analysis identified homozygosity for a splice donor mutation in intron 18. Exon trapping demonstrated that the mutation resulted in skipping of exon 18 and predicted deletion of 18 amino acids from the triple helical domain of the protein. In the second case, heterozygosity for a de novo 9 bp deletion in exon 40 of COL11A2 was identified, indicating that there are autosomal dominant forms of fibrochondrogenesis. These findings thus demonstrate that fibrochondrogenesis can result from either recessively- or dominantly-inherited mutations in COL11A2.
doi:10.1002/ajmg.a.34406
PMCID: PMC3264686  PMID: 22246659
Fibrochondrogenesis; skeletal dysplasia; COL11A2; collagen; cartilage
3.  Gain-of-function mutations in TRPV4 cause autosomal dominant brachyolmia 
Nature genetics  2008;40(8):999-1003.
The brachyolmias constitute a clinically and genetically heterogeneous group of skeletal dysplasias characterized by a short trunk, scoliosis and mild short stature1. Here, we identify a locus for an autosomal dominant form of brachyolmia on chromosome 12q24.1–12q24.2. Among the genes in the genetic interval, we selected TRPV4, which encodes a calcium permeable cation channel of the transient receptor potential (TRP) vanilloid family, as a candidate gene because of its cartilage-selective gene expression pattern. In two families with the phenotype, we identified point mutations in TRPV4 that encoded R616Q and V620I substitutions, respectively. Patch clamp studies of transfected HEK cells showed that both mutations resulted in a dramatic gain of function characterized by increased constitutive activity and elevated channel activation by either mechano-stimulation or agonist stimulation by arachidonic acid or the TRPV4-specific agonist 4α-phorbol 12,13-didecanoate (4αPDD). This study thus defines a previously unknown mechanism, activation of a calcium-permeable TRP ion channel, in skeletal dysplasia pathogenesis.
doi:10.1038/ng.166
PMCID: PMC3525077  PMID: 18587396
4.  The Shwachman–Bodian–Diamond syndrome gene mutations cause a neonatal form of spondylometaphysial dysplasia (SMD) resembling SMD Sedaghatian type 
Journal of Medical Genetics  2007;44(4):e73.
The Shwachman–Bodian–Diamond syndrome (SBDS) gene is a causative gene for Shwachman–Diamond syndrome, an autosomal recessive disorder with exocrine pancreatic insufficiency, bone marrow dysfunction and skeletal dysplasia. We report here on two patients with skeletal manifestations at the severest end of the phenotypic spectrum of SBDS mutations. An 11‐year‐old Japanese girl presented with neonatal respiratory failure necessitating lifelong ventilation support, severe short stature and severe developmental delay. She developed neutropenia in infancy, and decreased serum amylase was noted in childhood. A British boy was a stillbirth with pulmonary hypoplasia and hepatic fibrosis found on autopsy. Both cases had neonatal skeletal manifestations that included platyspondyly, lacy iliac crests and severe metaphysial dysplasia, and thus did not fall in the range of the known Shwachman–Diamond syndrome skeletal phenotype but resembled spondylometaphysial dysplasia (SMD) Sedaghatian type. The girl harboured a recurrent mutation (183TA→CT) and a novel missense mutation (79T→C), whereas the boy carried two recurrent mutations (183TA→CT and 258+2T→C). We also examined SBDS in one typical case with SMD Sedaghantian type and eight additional cases with neonatal SMD, but failed to discover SBDS mutations. Our experience expands the phenotypic spectrum of SBDS mutations, which, at its severest end, results in severe neonatal SMD.
doi:10.1136/jmg.2006.043869
PMCID: PMC2598034  PMID: 17400792
5.  A molecular and clinical study of Larsen syndrome caused by mutations in FLNB 
Journal of Medical Genetics  2006;44(2):89-98.
Background
Larsen syndrome is an autosomal dominant osteochondrodysplasia characterised by large‐joint dislocations and craniofacial anomalies. Recently, Larsen syndrome was shown to be caused by missense mutations or small inframe deletions in FLNB, encoding the cytoskeletal protein filamin B. To further delineate the molecular causes of Larsen syndrome, 20 probands with Larsen syndrome together with their affected relatives were evaluated for mutations in FLNB and their phenotypes studied.
Methods
Probands were screened for mutations in FLNB using a combination of denaturing high‐performance liquid chromatography, direct sequencing and restriction endonuclease digestion. Clinical and radiographical features of the patients were evaluated.
Results and discussion
The clinical signs most frequently associated with a FLNB mutation are the presence of supernumerary carpal and tarsal bones and short, broad, spatulate distal phalanges, particularly of the thumb. All individuals with Larsen syndrome‐associated FLNB mutations are heterozygous for either missense or small inframe deletions. Three mutations are recurrent, with one mutation, 5071G→A, observed in 6 of 20 subjects. The distribution of mutations within the FLNB gene is non‐random, with clusters of mutations leading to substitutions in the actin‐binding domain and filamin repeats 13–17 being the most common cause of Larsen syndrome. These findings collectively define autosomal dominant Larsen syndrome and demonstrate clustering of causative mutations in FLNB.
doi:10.1136/jmg.2006.043687
PMCID: PMC2598053  PMID: 16801345
6.  Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing 
BMC Genomics  2009;10:646.
Background
The emergence of next-generation sequencing technology presents tremendous opportunities to accelerate the discovery of rare variants or mutations that underlie human genetic disorders. Although the complete sequencing of the affected individuals' genomes would be the most powerful approach to finding such variants, the cost of such efforts make it impractical for routine use in disease gene research. In cases where candidate genes or loci can be defined by linkage, association, or phenotypic studies, the practical sequencing target can be made much smaller than the whole genome, and it becomes critical to have capture methods that can be used to purify the desired portion of the genome for shotgun short-read sequencing without biasing allelic representation or coverage. One major approach is array-based capture which relies on the ability to create a custom in-situ synthesized oligonucleotide microarray for use as a collection of hybridization capture probes. This approach is being used by our group and others routinely and we are continuing to improve its performance.
Results
Here, we provide a complete protocol optimized for large aggregate sequence intervals and demonstrate its utility with the capture of all predicted amino acid coding sequence from 3,038 human genes using 241,700 60-mer oligonucleotides. Further, we demonstrate two techniques by which the efficiency of the capture can be increased: by introducing a step to block cross hybridization mediated by common adapter sequences used in sequencing library construction, and by repeating the hybridization capture step. These improvements can boost the targeting efficiency to the point where over 85% of the mapped sequence reads fall within 100 bases of the targeted regions.
Conclusions
The complete protocol introduced in this paper enables researchers to perform practical capture experiments, and includes two novel methods for increasing the targeting efficiency. Coupled with the new massively parallel sequencing technologies, this provides a powerful approach to identifying disease-causing genetic variants that can be localized within the genome by traditional methods.
doi:10.1186/1471-2164-10-646
PMCID: PMC2808330  PMID: 20043857
7.  Disruption of the Flnb gene in mice phenocopies the human disease spondylocarpotarsal synostosis syndrome 
Human molecular genetics  2007;17(5):631-641.
Spondylocarpotarsal synostosis syndrome (SCT) is an autosomal recessive disease that is characterized by short stature, and fusions of the vertebrae and carpal and tarsal bones. SCT results from homozygosity or compound heterozygosity for nonsense mutations in FLNB. FLNB encodes filamin B, a multifunctional cytoplasmic protein that plays a critical role in skeletal development. Protein extracts derived from cells of SCT patients with nonsense mutations in FLNB did not contain filamin B, demonstrating that SCT results from absence of filamin B. To understand the role of filamin B in skeletal development, an Flnb−/− mouse model was generated. The Flnb−/− mice were phenotypically similar to individuals with SCT as they exhibited short stature and similar skeletal abnormalities. Newborn Flnb−/− mice had fusions between the neural arches of the vertebrae in the cervical and thoracic spine. At postnatal day 60, the vertebral fusions were more widespread and involved the vertebral bodies as well as the neural arches. In addition, fusions were seen in sternum and carpal bones. Analysis of the Flnb−/− mice phenotype showed that an absence of filamin B causes progressive vertebral fusions, which is contrary to the previous hypothesis that SCT results from failure of normal spinal segmentation. These findings suggest that spinal segmentation can occur normally in the absence of filamin B, but the protein is required for maintenance of intervertebral, carpal and sternal joints, and the joint fusion process commences antenatally.
doi:10.1093/hmg/ddm188
PMCID: PMC2680151  PMID: 17635842
8.  Cartilage-selective genes identified in genome-scale analysis of non-cartilage and cartilage gene expression 
BMC Genomics  2007;8:165.
Background
Cartilage plays a fundamental role in the development of the human skeleton. Early in embryogenesis, mesenchymal cells condense and differentiate into chondrocytes to shape the early skeleton. Subsequently, the cartilage anlagen differentiate to form the growth plates, which are responsible for linear bone growth, and the articular chondrocytes, which facilitate joint function. However, despite the multiplicity of roles of cartilage during human fetal life, surprisingly little is known about its transcriptome. To address this, a whole genome microarray expression profile was generated using RNA isolated from 18–22 week human distal femur fetal cartilage and compared with a database of control normal human tissues aggregated at UCLA, termed Celsius.
Results
161 cartilage-selective genes were identified, defined as genes significantly expressed in cartilage with low expression and little variation across a panel of 34 non-cartilage tissues. Among these 161 genes were cartilage-specific genes such as cartilage collagen genes and 25 genes which have been associated with skeletal phenotypes in humans and/or mice. Many of the other cartilage-selective genes do not have established roles in cartilage or are novel, unannotated genes. Quantitative RT-PCR confirmed the unique pattern of gene expression observed by microarray analysis.
Conclusion
Defining the gene expression pattern for cartilage has identified new genes that may contribute to human skeletogenesis as well as provided further candidate genes for skeletal dysplasias. The data suggest that fetal cartilage is a complex and transcriptionally active tissue and demonstrate that the set of genes selectively expressed in the tissue has been greatly underestimated.
doi:10.1186/1471-2164-8-165
PMCID: PMC1906768  PMID: 17565682
9.  WNT1 Mutations in Early-onset Osteoporosis and Osteogenesis Imperfecta 
The New England journal of medicine  2013;368(19):1809-1816.
SUMMARY
This report identifies human skeletal diseases associated with mutations in WNT1. In ten family members with dominantly inherited early-onset osteoporosis, a heterozygous missense variation c.652T>G (p.Cys218Gly) in WNT1 segregated with the disease, and a homozygous nonsense mutation (c.884C>A, p.Ser295*) was identified in two siblings with recessive osteogenesis imperfecta. In vitro, aberrant forms of WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. Wnt1 was clearly expressed in bone marrow, especially in B cell lineage and hematopoietic progenitors; lineage tracing identified expression in a subset of osteocytes, suggesting altered cross-talk of WNT signaling between hematopoietic and osteoblastic lineage cells in these diseases.
doi:10.1056/NEJMoa1215458
PMCID: PMC3709450  PMID: 23656646
10.  Mutations in SERPINF1 Cause Osteogenesis Imperfecta Type VI 
Abstract
Osteogenesis imperfecta (OI) is a spectrum of genetic disorders characterized by bone fragility. It is caused by dominant mutations affecting the synthesis and/or structure of type I procollagen or by recessively inherited mutations in genes responsible for the posttranslational processing/trafficking of type I procollagen. Recessive OI type VI is unique among OI types in that it is characterized by an increased amount of unmineralized osteoid, thereby suggesting a distinct disease mechanism. In a large consanguineous family with OI type VI, we performed homozygosity mapping and next-generation sequencing of the candidate gene region to isolate and identify the causative gene. We describe loss of function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF 1) in two affected members of this family and in an additional unrelated patient with OI type VI. SERPINF1 encodes pigment epithelium-derived factor. Hence, loss of pigment epithelium-derived factor function constitutes a novel mechanism for OI and shows its involvement in bone mineralization. © 2011 American Society for Bone and Mineral Research
doi:10.1002/jbmr.487
PMCID: PMC3214246  PMID: 21826736
BRITTLE BONE DISEASE; COLLAGEN TYPE I; FRACTURE; MATRIX PROTEINS; PIGMENT EPITHELIUM-DERIVED FACTOR
11.  Mutations in SERPINF1 Cause Osteogenesis Imperfecta Type VI 
Osteogenesis imperfecta (OI) is a spectrum of genetic disorders characterized by bone fragility. It is caused by dominant mutations affecting the synthesis and/or structure of type I procollagen or by recessively inherited mutations in genes responsible for the post-translational processing/trafficking of type I procollagen. Recessive OI type VI is unique among OI types in that it is characterized by an increased amount of unmineralized osteoid, thereby suggesting a distinct disease mechanism. In a large consanguineous family with OI type VI, we performed homozygosity mapping and next-generation sequencing of the candidate gene region to isolate and identify the causative gene. We describe loss of function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF1) in two affected members of this family and in an additional unrelated patient with OI type VI. SERPINF1 encodes pigment epithelium-derived factor. Hence, loss of pigment epithelium-derived factor function constitutes a novel mechanism for OI and demonstrates its involvement in bone mineralization.
doi:10.1002/jbmr.487
PMCID: PMC3214246  PMID: 21826736
Brittle bone disease; Collagen type I; Fracture; Matrix proteins; Pigment epithelium-derived factor
12.  Cartilage oligomeric matrix protein promotes cell attachment via two independent mechanisms involving CD47 and αVβ3 integrin 
Molecular and cellular biochemistry  2009;338(1-2):215-224.
Cartilage oligomeric matrix protein (COMP) is a pentameric ∼524 kDa multidomain extracellular matrix protein and is the fifth member of the thrombospondin family. COMP is abundantly expressed in proliferating and hypertrophic chondrocytes of the growth plate, articular cartilage, synovium, tendon, and ligament. The spatial localization of COMP highlights its importance in the phenotypes of pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), COMP disorders that are characterized by disproportionate short stature, brachydactyly, scoliosis, early-onset osteoarthritis, and joint hypermobility. In this study, the role of COMP in ligament was investigated with a series of cell attachment assays using ligament cells binding to COMP. A dose-dependent cell attachment activity was found, which was inhibited by a peptide containing the SFYVVMWK amino acid sequence derived from the globular C-terminal domain of COMP. This activity was independent of the recently described RGD-dependent attachment activity. Function-blocking antibodies to CD47 and αVβ3 integrin reduced cell attachment to COMP, implicating the participation of these cell surface molecules in COMP cell binding. Immunofluorescence studies showed that cell attachment to COMP induced the formation of lamellae containing F-actin microspikes associated with fascin. We propose that COMP promotes cell attachment via two independent mechanisms involving cell surface CD47 and αVβ3 integrin and that a consequence of cell attachment to COMP is the specific induction of fascin-stabilized actin microspikes.
doi:10.1007/s11010-009-0355-3
PMCID: PMC3150962  PMID: 20033473
COMP; Extracellular matrix; Cell adhesion; Signaling; Actin cytoskeleton
13.  Lethal Skeletal Dysplasia in Mice and Humans Lacking the Golgin GMAP-210 
The New England journal of medicine  2010;362(3):206-216.
BACKGROUND
Establishing the genetic basis of phenotypes such as skeletal dysplasia in model organisms can provide insights into biologic processes and their role in human disease.
METHODS
We screened mutagenized mice and observed a neonatal lethal skeletal dysplasia with an autosomal recessive pattern of inheritance. Through genetic mapping and positional cloning, we identified the causative mutation.
RESULTS
Affected mice had a nonsense mutation in the thyroid hormone receptor interactor 11 gene (Trip11), which encodes the Golgi microtubule-associated protein 210 (GMAP-210); the affected mice lacked this protein. Golgi architecture was disturbed in multiple tissues, including cartilage. Skeletal development was severely impaired, with chondrocytes showing swelling and stress in the endoplasmic reticulum, abnormal cellular differentiation, and increased cell death. Golgi-mediated glycosylation events were altered in fibroblasts and chondrocytes lacking GMAP-210, and these chondrocytes had intracellular accumulation of perlecan, an extracellular matrix protein, but not of type II collagen or aggrecan, two other extracellular matrix proteins. The similarities between the skeletal and cellular phenotypes in these mice and those in patients with achondrogenesis type 1A, a neonatal lethal form of skeletal dysplasia in humans, suggested that achondrogenesis type 1A may be caused by GMAP-210 deficiency. Sequence analysis revealed loss-of-function mutations in the 10 unrelated patients with achondrogenesis type 1A whom we studied.
CONCLUSIONS
GMAP-210 is required for the efficient glycosylation and cellular transport of multiple proteins. The identification of a mutation affecting GMAP-210 in mice, and then in humans, as the cause of a lethal skeletal dysplasia underscores the value of screening for abnormal phenotypes in model organisms and identifying the causative mutations.
doi:10.1056/NEJMoa0900158
PMCID: PMC3108191  PMID: 20089971
14.  Generalized Connective Tissue Disease in Crtap-/- Mouse 
PLoS ONE  2010;5(5):e10560.
Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB (coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in α1(I) and α1(II) chains, there was also loss of 3Hyp at proline 986 in α2(V) chains. In contrast, at two of the known 3Hyp sites in α1(IV) chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin.
doi:10.1371/journal.pone.0010560
PMCID: PMC2868021  PMID: 20485499
15.  CRTAP AND LEPRE1 MUTATIONS IN RECESSIVE OSTEOGENESIS IMPERFECTA 
Human mutation  2008;29(12):1435-1442.
Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Recently, dysregulation of hydroxylation of a single proline residue at position 986 of both the triple-helical domains of type I collagen α1(I) and type II collagen α1(II) chains has been implicated in the pathogenesis of recessive forms of OI. Two proteins, CRTAP, or cartilage-associated protein, and prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene) form a complex that performs the hydroxylation and brings the prolyl cis-trans isomerase cyclophilin-B (CYPB) to the unfolded collagen. In our screen of 78 subjects diagnosed with OI type II or III, we identified three probands with mutations in CRTAP and sixteen with mutations in LEPRE1. The latter group includes a mutation in patients from the Irish Traveller population, a genetically isolated community with increased incidence of OI. The clinical features resulting from CRTAP or LEPRE1 loss of function mutations were difficult to distinguish at birth. Infants in both groups had multiple fractures, decreased bone modeling (affecting especially the femurs), and extremely low bone mineral density. Interestingly, “popcorn” epiphyses may reflect underlying cartilaginous and bone dysplasia in this form of OI. These results expand the range of CRTAP/LEPRE1 mutations that result in recessive OI and emphasize the importance of distinguishing recurrence of severe OI of recessive inheritance from those that result from parental germline mosaicism for COL1A1 or COL1A2 mutations.
doi:10.1002/humu.20799
PMCID: PMC2671575  PMID: 18566967
Osteogenesis Imperfecta; Prolyl 3-Hydroxylation; CRTAP; LEPRE1

Results 1-15 (15)