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1.  Kuskokwim syndrome, a recessive congenital contracture disorder, extends the phenotype of FKBP10 mutations 
Human mutation  2013;34(9):1279-1288.
Recessive mutations in FKBP10 at 17q21.2, encoding FKBP65, cause both osteogenesis imperfecta (OI) and Bruck syndrome (OI plus congenital contractures). Contractures are a variable manifestation of null/missense FKBP10 mutations. Kuskokwim syndrome (KS) is an autosomal recessive congenital contracture disorder found among Yup’ik Eskimos. Linkage mapping of KS to chromosome 17q21, together with contractures as a feature of FKBP10 mutations, made FKBP10 a candidate gene. We identified a homozygous 3-nucleotide deletion in FKBP10 (c.877_879delTAC) in multiple Kuskokwim pedigrees; 3% of regional controls are carriers. The mutation deletes the highly conserved p.Tyr293 residue in FKBP65’s 3rd PPIase domain. FKBP10 transcripts are normal, but mutant FKBP65 is destabilized to a residual 5%. Collagen synthesized by KS fibroblasts has substantially decreased hydroxylation of the telopeptide lysine crucial for collagen cross-linking, with 2–10% hydroxylation in probands vs 60% in controls. Matrix deposited by KS fibroblasts has marked reduction in maturely cross-linked collagen. KS collagen is disorganized in matrix, and fibrils formed in vitro had subtle loosening of monomer packing. Our results imply that FKBP10 mutations affect collagen indirectly, by ablating FKBP65 support for collagen telopeptide hydroxylation by LH2, thus decreasing collagen crosslinks in tendon and bone matrix. FKBP10 mutations may also underlie other arthrogryposis syndromes.
PMCID: PMC3770534  PMID: 23712425
osteogenesis imperfecta; contractures; FKBP65; FKBP10; Bruck syndrome
2.  Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align With Collagen Binding Sites for Integrins and Proteoglycans 
Human mutation  2007;28(3):209-221.
Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proα1(I) and proα2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype–phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in α1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691–823 and 910–964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril–matrix interactions. Recurrences at the same site in α2(I) are generally concordant for outcome, unlike α1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In α2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype–phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events.
PMCID: PMC4144349  PMID: 17078022
osteogenesis imperfecta; type I collagen; genotype–phenotype; proteoglycan binding; COL1A1; COL1A2
3.  Absence of FKBP10 in Recessive Type XI Osteogenesis Imperfecta Leads to Diminished Collagen Cross-Linking and Reduced Collagen Deposition in Extracellular Matrix 
Human mutation  2012;33(11):1589-1598.
Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix.
PMCID: PMC3470738  PMID: 22718341
osteogenesis imperfecta; Bruck syndrome; FKBP65; FKBP10; PPIB; peptidylprolyl isomerase
4.  New Perspectives on Osteogenesis Imperfecta 
Nature reviews. Endocrinology  2011;7(9):540-557.
A new paradigm has emerged for osteogenesis imperfecta (OI) as a collagen-related disorder. The more prevalent autosomal dominant forms of OI are caused by primary defects in type I collagen, while autosomal recessive forms are caused by deficiency of proteins which interact with type I procollagen for post-translational modification and/or folding. Factors contributing to the mechanism of dominant OI include intracellular stress, disruption of interactions between collagen and non-collagenous proteins, compromised matrix structure, abnormal cell-cell and cell-matrix interactions and tissue mineralization. Recessive OI is caused by deficiency of any of the three components of the collagen prolyl 3-hydroxylation complex; absence of 3-hydroxylation is associated with increased modification of the collagen helix, supporting delayed collagen folding. Other causes of recessive OI include deficiency of collagen chaperones, FKBP65 or HSP47. Murine models are crucial to uncovering the common pathways in dominant and recessive OI bone dysplasia. Clinical management of OI is multidiscipinary, encompassing substantial progress in physical rehabilitation and surgical procedures, managment of hearing, dental and pulmonary abnormalities, as well as drugs such as bisphosphonates and rGH. Novel treatments using cell therapy or new drug regimens hold promise for the future.
PMCID: PMC3443407  PMID: 21670757
5.  A Founder Mutation in LEPRE1 Carried by 1.5% of West Africans and 0.4% of African Americans Causes Lethal Recessive Osteogenesis Imperfecta 
Genetics in Medicine  2012;14(5):543-551.
Deficiency of prolyl 3-hydroxylase 1, encoded by LEPRE1, causes recessive osteogenesis imperfecta. We previously identified a LEPRE1 mutation, exclusively in African Americans and contemporary West Africans. We hypothesized that this allele originated in West Africa and was introduced to the Americas with the Atlantic slave trade. We aimed to determine the frequency of carriers for this mutation among African Americans and West Africans, and the mutation origin and age.
Genomic DNA was screened for the mutation using PCR and restriction digestion, and a custom TaqMan genomic SNP assay. The mutation age was estimated using microsatellites and short tandem repeats spanning 4.2 Mb surrounding LEPRE1 in probands and carriers.
Approximately 0.4% of Mid-Atlantic African Americans carry this mutation, estimating recessive OI in 1/260,000 births in this population. In Nigeria and Ghana, 1.48% of unrelated individuals are heterozygous carriers, predicting 1/18,260 births will be affected with recessive OI, equal to the incidence of de novo dominant OI. The mutation was not detected in Africans from surrounding countries. All carriers shared a haplotype of 63-770 Kb, consistent with a single founder for this mutation. Using linkage disequilibrium analysis, the mutation was estimated to have originated between 650 and 900 years before present (1100-1350 C.E.).
We identified a West African founder mutation for recessive OI in LEPRE1. Nearly 1.5% of Ghanians and Nigerians are carriers. The age of this allele is consistent with introduction to North America via the Atlantic slave trade (1501 – 1867 C.E).
PMCID: PMC3393768  PMID: 22281939
LEPRE1; osteogenesis imperfecta; founder mutation; West Africa
6.  COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta 
Human mutation  2011;32(6):598-609.
Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.
PMCID: PMC3103631  PMID: 21344539
Osteogenesis imperfecta; C-propeptide; collagen; C-proteinase; mineralization; high bone mass
7.  A Novel Mutation in LEPRE1 That Eliminates Only the KDEL ER- Retrieval Sequence Causes Non-Lethal Osteogenesis Imperfecta 
PLoS ONE  2012;7(5):e36809.
Prolyl 3-hydroxylase 1 (P3H1), encoded by the LEPRE1 gene, forms a molecular complex with cartilage-associated protein (CRTAP) and cyclophilin B (encoded by PPIB) in the endoplasmic reticulum (ER). This complex is responsible for one step in collagen post-translational modification, the prolyl 3-hydroxylation of specific proline residues, specifically α1(I) Pro986. P3H1 provides the enzymatic activity of the complex and has a Lys-Asp-Glu-Leu (KDEL) ER-retrieval sequence at the carboxyl terminus. Loss of function mutations in LEPRE1 lead to the Pro986 residue remaining unmodified and lead to slow folding and excessive helical post-translational modification of type I collagen, which is seen in both dominant and recessive osteogenesis imperfecta (OI). Here, we present the case of siblings with non-lethal OI due to novel compound heterozygous mutations in LEPRE1 (c.484delG and c.2155dupC). The results of RNA analysis and real-time PCR suggest that mRNA with c.2155dupC escapes from nonsense-mediated RNA decay. Without the KDEL ER- retrieval sequence, the product of the c.2155dupC variant cannot be retained in the ER. This is the first report of a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval sequence, whereas other functional domains remain intact. Our study shows, for the first time, that the KDEL ER- retrieval sequence is essential for P3H1 functionality and that a defect in KDEL is sufficient for disease onset.
PMCID: PMC3352923  PMID: 22615817
8.  Null mutations in LEPRE1 and CRTAP cause severe recessive osteogenesis imperfecta 
Cell and tissue research  2009;339(1):59-70.
Classical osteogenesis imperfecta (OI) is a dominant genetic disorder of connective tissue caused by mutations in either of the two genes encoding type I collagen, COL1A1 and COL1A2. Recent investigations, however, have generated a new paradigm for OI incorporating many of the prototypical features that distinguish dominant and recessive conditions, within a type I collagen framework. We and others have shown that the long-sought cause of the recessive form of OI, first postulated in the Sillence classification, lies in defects in the genes encoding cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1/LEPRE1). Together with cyclophilin B (PPIB), CRTAP and P3H1 comprise the collagen prolyl 3-hydroxylation complex, which catalyzes a specific post-translational modification of types I, II, and V collagen, and may act as a general chaperone. Patients with mutations in CRTAP or LEPRE1 have a lethal to severe osteochondrodystrophy that overlaps with Sillence types II and III OI but has distinctive features. Infants with recessive OI have white sclerae, undertubulation of the long bones, gracile ribs without beading, and a small to normal head circumference. Those who survive to childhood or the teen years have severe growth deficiency and extreme bone fragility. Most causative mutations result in null alleles, with the absence or severe reduction of gene transcripts and proteins. As expected, 3-hydroxylation of the Pro986 residue is absent or severly reduced, but bone severity and survival length do not correlate with the extent of residual hydroxylation. Surprisingly, the collagen produced by cells with an absence of Pro986 hydroxylation has helical overmodification by lysyl hydroxylase and prolyl 4-hydroxylase, indicating that the folding of the collagen helix has been substantially delayed.
PMCID: PMC3156555  PMID: 19862557
Recessive osteogenesis imperfecta; CRTAP; P3H1/LEPRE1; Prolyl 3-hydroxylation; Osteochondrodysplasia
9.  Lack of Cyclophilin B in Osteogenesis Imperfecta with Normal Collagen Folding 
The New England journal of medicine  2010;362(6):521-528.
Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband’s collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis–trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought.
PMCID: PMC3156560  PMID: 20089953
10.  Prolyl 3-hydroxylase 1 and CRTAP are mutually stabilizing in the endoplasmic reticulum collagen prolyl 3-hydroxylation complex 
Human Molecular Genetics  2009;19(2):223-234.
Null mutations in cartilage-associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1/LEPRE1) cause types VII and VIII OI, respectively, two novel recessive forms of osteogenesis imperfecta (OI) with severe to lethal bone dysplasia and overmodification of the type I collagen helical region. CRTAP and P3H1 form a complex with cyclophilin B (CyPB) in the endoplasmic reticulum (ER) which 3-hydroxylates the Pro986 residue of α1(I) and α1(II) collagen chains. We investigated the interaction of complex components in fibroblasts from types VII and VIII OI patients. Both CRTAP and P3H1 are absent or reduced on western blots and by immunofluorescence microscopy in cells containing null mutations in either gene. Levels of LEPRE1 or CRTAP transcripts, however, are normal in CRTAP- or LEPRE1-null cells, respectively. Stable transfection of a CRTAP or LEPRE1 expression construct into cells with null mutations for the transfected cDNA restored both CRTAP and P3H1 protein levels. Normalization of collagen helical modification in transfected CRTAP-null cells demonstrated that the restored proteins functioned effectively as a complex. These data indicate that CRTAP and P3H1 are mutually stabilized in the collagen prolyl 3-hydroxylation complex. CyPB levels were unaffected by mutations in either CRTAP or LEPRE1. Proteasomal inhibitors partially rescue P3H1 protein in CRTAP-null cells. In LEPRE1-null cells, secretion of CRTAP is increased compared with control cells and accounts for 15–20% of the decreased CRTAP detected in cells. Thus, mutual stabilization of P3H1 and CRTAP in the ER collagen modification complex is an underlying mechanism for the overlapping phenotype of types VII and VIII OI.
PMCID: PMC2796888  PMID: 19846465

Results 1-10 (10)